AN ABSTRACT OF THE THESIS OF
Ana L. Scopel
for the degree of
Doctor of Philosophy
in
Crop Science presented on July 23, 1993.
Title:
Photocontrol of geed Germination in Arable Land
Abstract approved:
Redacted for Privacy
Steven R. Radosevich
The overall objective of this thesis was to determine
the role of light in triggering germination during soil
disturbance, and to identify the mechanisms whereby buried
seeds in arable soils detect the light signals.
In the first section it is shown that a short period
of burial induces a dramatic, m 10.000-fold increase in
light sensitivity in the seeds of the arable weed Datura
ferox. This increase in sensitivity has been interpreted as
a natural transition from the "low-fluence" (LF) to the
"very-low-fluence" (VLF) mode of phytochrome action. Field
experiments indicated that germination of buried seeds may
be triggered by millisecond-exposures to sunlight. This
observation suggests a key role for the process of
sensitization in the mechanisms that allows seeds to detect
soil cultivation events in arable lands.
Large scale field experiments using standard farm
equipment further tested the above hypothesis. Results
demonstrated that cultivating agricultural land during
daytime can increase germination of buried seed populations
between 70 and 400 t above levels recorded following
nighttime cultivations. Covering the tillage implements
during daytime cultivation decreased the number of
dicotyledonous seedlings emerged, while strong artificial
illumination (> 300 Amol m-2 5-1; 400-800 nm) of the soil
surface during nighttime tillage significantly increased
seedling densities. These results suggest that the
enhancement of seed germination caused by daytime tillage,
compared with nighttime tillage, is due to light that
penetrates into the soil during the actual disturbance
event. The detection by the seeds of the extremely short
exposure to sunlight requires high photosensitivity, and
provides an "adaptive purpose" for the evolution of the VLF
response mechanism in phytochrome-controlled seed
germination.
Seeds of Datura ferox were used to investigate the
connection between the induction of sensitivity to VLFs and
endogenous abscisic acid (ABA) levels. Results indicated
that, in buried seeds, the decline in endogenous ABA levels
coincided with or preceded the induction of the VLF
germination response. Exogenous ABA applied to the
incubation medium preferentially reduced the VLF response
compared with its effect on the LF response. These results
support the hypothesis that the reduction in endogenous ABA
levels during seed burial plays a role in switching the
seeds from the LF to the VLF mode of phytochrome action.
Photocontrol of Seed Germination in Arable Land
by
Ana L. Scopel
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed July 23, 1993
Commencement June 1994
APPROVED:
Redacted for Privacy
Pro essor of Crop and Soil Science in charge of major
Redacted for Privacy
H6ad of department of Crop and Soil Science
Redacted for Privacy
Dean of G
Date thesis is presented July 23, 1993
Typed by
Ana L Scopel
ACKNOWLEDGEMENTS
I specially appreciate the support and guidance that
Steve Radosevich has given to me in all the aspects related
with my work at OSU. The opportunity of discussing and
sharing different views about science, society and ecology
with him and Mary Lynn Roush has been most enriching, and
has certainly contributed to modify my "worldview". I
enjoyed working with Anita Azarenko and specially value what
I have learned about hormone analysis. I would also like to
thank Mayvin Sinclair, for her friendship and support, and
Fred Friedow for being always so helpful in solving
problems.
I thank the Consejo Nacional de Investigaciones
Cientificas y Tecnicas (CONICET) of Argentina for granting
me an External Fellowship that made this visit to the United
States possible and to the Dept. of Forest Science, OSU, for
additional financial support.
Finally my gratitude to my husband, Carlos, for his
love, unconditional support and encouragement, and to my two
dearest treasures, my sons, Nicolas and Sebastian. To them,
and my family at home is this thesis dedicated.
CONTRIBUTION OF AUTHORS
Co-authors of individual papers are identified in the
first page of each chapter. The following is brief list of
their contributions to each manuscript.
Anita N. Azarenko advised me on techniques used for
ABA quantitation and collaborated in the design and
execution of the experiments presented in chapter 4,
contributed to the discussion of the results, and edited
successive drafts of the paper.
Carlos L. Ballare contributed to the ideas and
hypotheses discussed in chapters 2, 3, and 4, collaborated
during the execution of the experiments, and reviewed drafts
of the manuscripts.
Steven R. Radosevich advised me on several steps along
the completion of this thesis, contributed to the ideas
discussed in this work, to the discussion of the
experimental results, and edited successive drafts of the
thesis chapters.
Rodolfo A. Sanchez discussed with me several of the
ideas presented in chapter 2 and 4, contributed to the
discussion of the results, and edited drafts of the
manuscript included in chapter 2.
TABLE OF CONTENTS
CHAPTER 1. GENERAL INTRODUCTION
References
CHAPTER 2. INDUCTION OF EXTREME LIGHT SENSITIVITY
IN BURIED WEED SEEDS AND ITS ROLE IN THE PERCEPTION
OF SOIL CULTIVATIONS
Abstract
Introduction
Materials and Methods
Results and Discussion
Conclusions
References
CHAPTER 3. PHOTOSTIMULATION OF SEED GERMINATION
DURING SOIL TILLAGE
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusions
References
1
13
22
23
24
27
33
40
41
52
53
55
60
67
72
78
80
CHAPTER 4. ABSCISIC ACID LEVELS AND THE INDUCTION
OF THE VERY-LOW-FLUENCE RESPONSE IN PHYTOCHROME
CONTROLLED SEED GERMINATION
Abstract
Introduction
Materials and methods
Results
Discussion
References
90
91
93
97
102
106
110
CHAPTER 5. SUMMARY AND OUTLOOK
121
BIBLIOGRAPHY
126
APPENDICES
Apendix 1. Statistical analyses of data
presented in Chapter 3
Appendix 2. Statistical analyses of data
presented in Chapter 4
140
140
152
LIST OF FIGURES
Figure 2.1 (A) Light guides used to irradiate buried
seeds with sunlight in the field. (B) Mount
used to held the seed coats during
determinations of spectral transmittance
48
Figure 2.2 (A) Relative spectral photon distribution
(LI 1800
of the treatment light sources
spectroradiometer, Li-Cor, NE, U.S.A.).
(B) Transmittance spectra of the seed coats
of D. ferox. (C) Relationships between
germination in the laboratory and the W Pfr
established by R and FR light
49
Figure 2.3 Fluence response curves for the induction
of germination by sunlight under field
conditions
50
Figure 2.4 (A) Calculated Pfr/P ratios established by
different fluences of sunlight.
(B) Relationship between germination and the
W Pfr established by the various exposures
to sunlight or sunlight followed by FR
51
Figure 3.1 Effect of daytime cultivation in early summer
(1991) on seedling emergence compared with the
86
the nighttime (no light) control
Figure 3.2 Effect of daytime cultivation in late
summer (1991) on seedling emergence compared
with the nighttime (no light) control
87
Figure 3.3 Effect of covering the tillage implements
during daytime cultivation in the autumn
(1991) on seedling emergence compared with
the full sunlight (no cover) control
88
Figure 3.4 Effect of manipulating the light conditions
during cultivation on the emergence of
dicotyledonous seedlings compared with
the nighttime (no light) control
89
Figure 4.1 Total (upper panel) and selected ion
chromatograms of methylated HPLC
purified sample of ABA from intact seeds
of D. ferox
115
Figure 4.2 Time courses of dark germination, light
sensitivity and ABA levels in D. ferox
seeds buried outdoors
116
Figure 4.3 Time courses of dark germination, light
sensitivity and ABA levels in D. ferox
seeds buried in a growth chamber at
20°C/30°C.
117
Figure 4.4 Effect of exogenous ABA on light
sensitivity of D. ferox seeds exhumed
after five (A) or six (B) months of burial
outdoors
118
Figure 4.5 Effect of exogenous ABA on light
sensitivity of D. ferox seeds that had
been kept for three weeks in a WSA
119
Figure 4.6 Relationships between VLF responsivity
and endogenous ABA levels in whole
seeds of D. ferox
120
LIST OF TABLES
Table 2.1
Table 3.1
Viability and germination of different seed
batches of Datura ferox at the time of burial
(late winter)
47
Cultivation experiments carried out during
1991 and 1992
85
PHOTOCONTROL OF SEED GERMINATION IN ARABLE LAND
CHAPTER 1
GENERAL INTRODUCTION
"But seeds are invisible. They sleep deep in the heart of
the earth's darkness, until some one among them is seized
with the desire to awaken."
Antoine de Saint Exupery,
The Little Prince
Harcourt, Brace & World Inc.
2
Seed banks in arable land
Buried seed populations ("seed banks") are recognized
as a primary source of new plants of annual species that
occupy arable land. Efforts have been directed towards the
reduction of seed banks by crop rotations, fallowing and
other management methods involving cultivation (for review
see Cavers and Benoit, 1989). Scheweizer and Zimdahl (1984),
summarizing 50-year crop management studies in experimental
areas, found that, regardless of the agricultural practice
used, most viable seeds were lost from the seed bank within
1-4 years if the input of weed seeds was prevented or was
minimal. Norris (1985 cited in Menges 1987) estimated that
weed control must exceed 99.99 % efficiency in order to
reduce seed inputs to mantain a constant seed bank in an
arable field. The results obtained by Ballare et al.
(1987)
when modelling the dynamics of Datura ferox populations in
soybean crops support these estimations. These authors found
that control efficiencies as large as 95 % could not prevent
the population of this weed from increasing year by year.
Farmers continue to be dependent on herbicide use to
control the input of weed seeds and also to meet the
standards imposed by the goverment and retail sales, such as
crop seed free of contaminants or high cosmetic standards
for fresh market produce. As a standard practice in the
3
U.S.A., any land in which a crop is grown undergoes at least
one major soil disturbance (e.g. ploughing) and from 2 to 6
subsequent tillages (e.g. a disk + roller) for seed bed
preparation. A prevalent strategy for weed control consists
of preplant and preemergent herbicides or 3 to 5
cultivations after crop emergence. These practices are not
only expensive in terms of herbicide cost, human labor and
fuel but also can have a negative effect on soil structure.
Frisbie and Smith (1989) while addressing the future
of agriculture propose that bio-intensive integrated pest
management will rely on biological control, host resistance
and cultural management as the main tactics for crop
production rather than pesticide-dependant methods. They
recognize the need for a creative scientific revolution to
take place, especially in the area of weed control.
nHowever, new insights into how to create a significant
change in current weed control procedures will only be
brought about through a thorough understanding of plant
environmental biology. For example, possible alternatives to
herbicide application might be achieved by manipulating
microenvironmental stimuli important for weed seed
germination and seedling emergence.
4
Environmental control of germination in arable land
Successional environments are characterized by
transient increases in the availability of light and other
environmental resources, which are caused by disturbances
that eliminate or reduce existing vegetation. Canopy "gaps"
may be created by natural agents (i.e. fire, storms, or
herbivores) or by humans in managed ecosystems. Gaps may be
of different sizes, from minute ones created by small
herbivores foraging on herbaceous vegetation to those
involving hundreds of hectares caused by tillages in
cultivated areas.
In arable lands, disturbances occur in a more or less
cyclical fashion due to the practices necessary to produce a
particular crop. The major disturbances in such agricultural
systems are grazing, mowing, and soil cultivation. Soil
cultivation in arable land usually results in a cohort
(flush) of weed seedling emergence and recruitment (e.g.
Chancellor, 1964; Roberts and Potter, 1980; Froud-Williams
et al., 1984). Recruitment of some weed species takes place
almost exclusively after tillage, suggesting a sophisticated
degree of adjustment to the agricultural environment
(Soriano et al., 1970; Harper, 1977; Ballare et al., 1992).
Several factors have been proposed to work as
5
environmental signals whereby seeds detect disturbances and
canopy gaps. These include soil temperature changes (Koller,
1972; Thomson et al., 1977; Aldrich, 1984; Benech Arnold et
al., 1988; Ghersa et al., 1993), light (Sauer and Struik,
1964; Wesson and Wearing, 1969), changes in the soil
atmosphere (Roberts, 1962; Simmonds and Simpson, 1971;
Harper, 1977), and modification of chemical composition of
the soil solution (Angevine and Chabot, 1979; Pons, 1989;
Simpson, 1990). The role of light as a stimulus for
germination in disturbed soil has become well established
since the classic experiments of Sauer and Struick (1964)
and Wesson and Wareing (1969). Although these authors did
not cultivate the soil with tillage equipment in their
experiments, they convincingly demonstrated that germination
in disturbed soil can be reduced if light is excluded.
Different authors have subsequently focused on changes in
light sensitivity during seed burial (e.g. Taylorson, 1972;
Baskin and Baskin, 1979, 1980; and refs. in Karssen, 1982)
and the involvement of phytochrome in the germination
responses to light (see Taylorson, 1987 and Ballare et al.,
1992 for reviews).
Phytochrome
The discovery of phytochrome in the
early 1950s
(Borthwick et al., 1952) led to a tremendous increase in
6
understanding about how plants perceive and respond to light
(see Sage, 1992 for a historical account). Since phytochrome
is the photoreceptor involved in the photocontrol of seed
germination a brief overview of its major properties
follows. More detailed treatment of the subject can be found
in several recent books (e.g. Kronenberg and Kendrick Eds.,
1986; Furuya Ed., 1987; Attridge, 1990).
It is now known that phytochrome is not a single
pigment, but rather a family of proteins with a similar,
perhaps identical chromophore (Sharrock and Quail, 1989;
Furuya, 1989; Quail, 1991). In Arabidopsis for example,
three different phytochromes and five different phytochrome
genes have been discovered over the last few years.
In the mechanism of action of these photoreceptors the
first step involved is absorption of light by the
chromophore, which is accompanied by a change in the
properties of the pigment molecule. This alteration
initiates a transduction process, that is a series of
molecular events that culminate in the response, e.g.
germination. The precise sequence of events is not yet known
for any particular response, but the gap in understanding of
how the "black boxes" work between light absorption and
observable response has been dramatically narrowed during
the last few years. Phytochrome-mediated processes are being
7
studied intensively. Of general acceptance is that there is
more than just one primary mechanism of phytochrome action
(see e.g. Johnson, 1989). In some cases it is clear that
phytochrome responses do not involve changes in gene
expression (e.g. chloroplast orientation in the alga
Mougeotia). Yet, in others (probably the majority of
phytochrome responses) light absorption by phytochrome leads
to altered expression of selected genes. There is little
evidence for a direct interaction between phytochrome and
the genome and, possibly, there is more than one mechanism
whereby phytochromes effect transcriptional activity (see
Quail, 1991 for a review).
Regarding its photochemical characteristics,
phytochrome molecules exist in two relatively stable forms,
Pr (active form) and Pfr, with absorption maxima in the red
(R) and far-red (FR) regions of the electromagnetic
spectrum, respectively. Each form is converted into the
other upon absorption of light. Because the absorption
spectra of Pr and Pfr overlap to a considerable extent,
light cannot push the pigment to pure Pr or Pfr. Thus, under
conditions of continuous illumination, a photoequilibrium
(0) between Pr and Pfr is reached that may be characterized
by the proportion of phytochrome molecules that are in the
Pfr form, i.e. the Pfr/P ratio. Values of 0 vary from 0.02
under monochromatic FR to 0.86 under monochromatic R (e.g.
8
Mancinelli, 1988). If the pigment is exposed to
polychromatic light, as is the case under natural
conditions, the Pfr/P ratio at 0 is highly dependent on the
R to FR quantum ratio in the incident radiation (R:FR ratio)
(Smith and Holmes, 1977). Thus, 0 values obtained by
exposing purified phytochrome preparations to natural light
typically vary from ca. 0.6 (direct sunlight; R:FR = 1.15)
to 0.15 (light filtered through dense leaf canopy; R:FR =
0.2). The effects of light on a number of physiological
functions are well correlated with its effects on 0. Values
of 0 can be estimated analytically (Hartmann and Cohnen-
Unser, 1972) using spectral irradiance data and published
values of phytochrome photoconversion-coefficients (e.g.
Mancinelli, 1988). A similar approach can be used to
calculate the rate of cycling between Pr and Pfr, and to
estimate the amount of Pfr formed after exposures to light
that are too short to drive the pigment to photoequilibrium
(see Cone and Kendrick, 1985; Scopel et al., 1991).
Photocontrol of seed germination
Laboratory studies on the photocontrol of germination
have shown that light must establish a relatively high level
of the active form of phytochrome (Pfr) for germination to
occur, and that the potential effect of light can be
nullified if Pfr is reduced to about 2 percent with a pulse
9
of far-red radiation (see references in Bewley and Black,
1982). This FR-reversible photoresponse is known as the low
fluence (LF) response. Recent work in several laboratories
has demonstrated that many phytochrome mediated processes
can be elicited by light exposures that establish much lower
percentages of Pfr. This type of photoresponse, termed very
low fluence (VLF) response (Mandoli and Briggs, 1981;
Blaauw-Jansen, 1983; Kronenberg and Kendrick, 1986), also
has been documented for light promoted seed germination. For
example, sensitivity of seeds to light forming very low
amounts of Pfr has been obtained in laboratory experiments
by pretreatments of chilling (VanDerWoude and Toole, 1980;
Cone et al., 1985; VanDerWoude, 1985), high temperature
incubation (Cone et al., 1985; Taylorson and Dinola, 1989),
gibberellic acid (Rethy et al., 1987), or ethanol
(VanDerWoude, 1985).
Sensitized seeds require ca. 104 fewer
photons than unsensitized seeds, and can be induced to
germinate by Pfr levels that are two to four orders of
magnitude lower than those established by a saturating pulse
of pure far-red radiation. The discovery of VLF response in
seeds has prompted a consideration of its potential
ecological significance (Cone et al., 1985). Taylorson
(1972) and Baskin and Baskin (1979), working with seed
recovered from soil, reported levels of light sensitivity
that were difficult to interpret based on classical far-red
reversible phytochrome responses. These findings suggested
10
that the VLF response mechanism may be operative in buried
seeds.
An assessment of the ecological importance of light
sensitization, i.e. VLF response, requires that light
conditions encountered by seeds in arable soil be
considered.
If seeds are buried deeper than a few mm, they
will be exposed to darkness (Woolley and Stoller, 1978;
Tester and Morris, 1987) for relatively long periods of
time, and eventually, to pulse-like irradiations of sunlight
when the soil is cultivated. Thereafter, only a fraction of
the total seeds in the soil should experience further light,
because light penetrates so poorly into soil. Moreover,
seeds on the soil surface are at great risk from
desiccation, photoinhibition (Gorski and Garska, 1979), and
predation (Scopel et al., 1988). Therefore, if light is an
important cue for germination in cultivated soil,
germinating cohorts following soil disturbance would seem
most dependent on seeds being exposed to light during
tillage. Studies by Hartmann and Nezadal (1990) support this
reasoning. These scientists, using a farm-scale version of
the pioneering experiment of Sauer and Struik (1964),
observed that weed seedling emergence in arable fields was
strongly reduced when tillage operations were performed at
night.
11
If the VLF response mechanism is indeed found in
buried seeds, the possibility of it being a general
phenomena among light-requiring seeds should be further
investigated. Although the evidence reviewed above suggests
a role for light in triggering germination during tillage,
no direct proof for this hypothesis has been obtained using
normal cultivation conditions. Moreover, the contribution of
light received during the actual disturbance in relation to
the total light received by seeds in tilled soil has never
been evaluated. The physiological basis for the shift to the
VLF response also is unknown (Cone et al., 1985;
VanderWoude, 1985). The endogenous balance of growth
regulators might be involved in seeds because, under
laboratory conditions, experiments with increases
in gibberellic acid concentration sometimes cause seeds to
respond within the VLF range (Rethy et al., 1987).
This thesis is focused on the photocontrol of seed
germination in arable lands. Each chapter was written as an
individual manuscript and can be read independently of the
others.
In the subsequent chapter (Chapter 2), the main
question addressed is whether seeds shift during burial from
the LF response mode of phytochrome action (in which only a
relatively high % Pfr can trigger germination), to the VLF
12
response (in which seeds may be induced to germinate by much
lower % Pfr (< 10-2U. In Chapter 3, the ecological
significance of VLF responses is investigated in relation to
the perception of light by the buried seeds during soil
disturbance. In Chapter 4, the involvement of abscicic acid
in controlling the shift from the LF response to the VLF
response is investigated. Finally, Chapter 5 provides a
synthesis of the major results presented in this thesis.
13
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Scopel AL, Ballard CL, Sanchez RA (1991) Induction of
extreme light sensitivity in buried weed seeds and its
role in the perception of soil cultivations. Plant Cell
and Environment 14: 501-508
Sharrock RA, Quail PH (1989) Novel phytochrome sequences in
Arabidopsis thaliana: structure, evolution, and
differential expression of a plant regulatory
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photoreceptor family. Genes and Development 3: 1745­
1757
Schweizer EE, Zimdahl RL (1984) Weed seed decline in
irrigated soil after six years of continuous corn (Zea
mays) and herbicides. Weed Science 32: 76-83
Simmonds JA, Simpson GM (1971) Canadian Journal of Botany
49: 1833-1839
Simpson GM (1990) Seed dormancy in grasses. Cambridge
University Press, Cambridge, UK
Smith H, Holmes MG (1977) The function of phytochrome in the
natural environment -III. Measurement and calculation
of phytochrome photoequilibria. Photochemistry and
Photobiology 25: 547-550
Soriano A, De Eilberg BA, Suero A (1970) Effects of burial
and changes of depth in the soil on seeds of Datura
ferox L. Weed Research 11: 196-199
Taylorson RB (1972) Phytochrome controlled changes in
dormancy and germination of buried weed seeds. Weed
Science 20: 417-422
Taylorson RB (1987) Environmental and chemical manipualtion
of weed seed dormancy. Reviews in Weed Science 3: 135­
154
Taylorson RB, Dinola L (1989) Increased phytochrome
responsiveness and a high-temperature transition in
Barnyardgrass (Echinochloa crus-galli) seed dormancy.
Weed Science 37: 335-338
20
Tester M, Morris C (1987) The penetration of light through
the soil. Plant Cell and Environment 10: 281-286
Thompson K (1987) Seeds and seed banks. New Phytologist 106
(Suppl.): 23-34
Thompson K, Grime JP, Mason G (1977) Seed germination in
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267: 147-148
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enhancement of phytochrome-dependent lettuce seed
germination by prechilling. Plant Physiology 66: 220­
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VanderWoude WJ (1985) A dimeric mechanism for the action of
phytochrome: evidence from photothermal interactions in
lettuce seed germination. Photochemistry and
Photobiology 42: 655-661
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181-190
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21
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22
CHAPTER 2
INDUCTION OF EXTREME LIGHT SENSITIVITY IN BURIED WEED SEEDS
AND ITS ROLE IN THE PERCEPTION OF SOIL CULTIVATIONS
Ana L. Scopel, Carlos L. Ballare and Rodolfo A. Sanchez
Departamento de Ecologia, Facultad de Agronomia,
University of Buenos Aires,
(1417) Buenos Aires, Argentina
(A.L.S., C.L.B., R.A.S.); Departments of Forest Science and
Crop and Soil Science, Oregon State University, Corvallis,
OR 97331-7501, USA (A.L.S., C.L.B.)
Plant, Cell and Environment 14: 501-508 (1991)
23
ABSTRACT
Light, probably acting through the photoreceptor
phytochrome, promotes germination of weed seeds when the
soil is disturbed by tillage operations. A short period of
burial is shown to induce an enormous, . 10,000-fold
increase in light sensitivity in the seeds of the arable
weed Datura ferox which is interpreted as being a natural
transition to the "very-low-fluence" mode of phytochrome
action. Field experiments indicated that germination of
buried seeds may be triggered by millisecond-exposures to
sunlight and suggested a key role for the process of
sensitization in the mechanisms whereby light requiring
seeds detect the occurrence of soil cultivation events in
arable lands.
Abbreviations: FR, far-red; LFR, low fluence response; P,
total phytochrome (Pfr + Pr); Pfr, FR-absorbing form of
phytochrome; Pr, R-absorbing form of phytochrome; R, red;
VLFR, very low fluence response; 0, Pfr/Pr ratio at
photoequilibrium; ai,x
and a2,),
apparent spectral molar
conversion cross-sections for the transitions Pr -> Pfr and
Pfr -> Pr, respectively.
Key words: Datura ferox; germination ecology; phytochrome;
seed dormancy; soil cultivation; very low fluence; weeds.
24
INTRODUCTION
The ability of terrestrial plants to produce dormant
seeds presumably evolved more than 300 million years ago
(Mapes et al., 1989), and is of indubitable value for
reducing germination under environmental conditions that are
unfavourable for seedling establishment. In a number of
species, including many weeds of arable land, dormancy is
terminated when the hydrated seed is exposed to light, which
is perceived by the photoreversible chromoprotein
phytochrome (e.g. Bewley and Black, 1982; Taylorson, 1987).
Frequently, in order to be effective, light must establish a
relatively high level of the far-red (FR) absorbing form of
phytochrome (Pfr), and its potential effect can be
completely nullified if the Pfr content is immediately
reduced to . 2% with a pulse of FR radiation. This FR-
reversible photoresponse, known as the "low fluence response
(LFR)" (Kronenberg and Kendrick, 1986), has generated
considerable interest because of its involvement in the
regulation of seed germination under leaf canopies (Cumming,
1963; Smith, 1973; Frankland and Taylorson, 1983).
In recent years, work in several laboratories has
shown that certain phytochrome-mediated processes can be
elicited by light treatments that establish much lower
percentage of Pfr. This type of photoresponse, termed "very
25
low fluence response (VLFR)" (Mandoli and Briggs, 1981;
Blaauw-Jansen, 1983; Kronenberg and Kendrick, 1986), has
also been documented for light promoted seed germination.
Light sensitivity of seeds of some species that form very
low amounts of Pfr has been obtained in the laboratory by
pretreatments such as chilling (VanDerWoude and Toole, 1980;
Cone et al., 1985; VanDerWoude, 1985), incubation at high
temperatures (Cone et al., 1985; Taylorson and Dinola,
1989), incubation in the presence of gibberellic acid (Rethy
et al., 1987), or treatment with anesthetics (VanDerWoude,
1985). Sensitive seeds require 104 times less photons than
insensitive seeds and can be induced to germinate by Pfr
levels that are two to four orders of magnitude lower than
those established by a saturating pulse of pure FR.
The discovery of the VLFR in seeds has prompted a
number of questions about its potential ecological
significance (Cone et al., 1985). As yet, little is known
about the occurrence and adaptive implications of this type
of light-controlled germination under natural conditions.
Seeds are known to undergo important changes in light
sensitivity during burial (Taylorson, 1970, 1972; Baskin and
Baskin, 1980, 1985; Froud-Williams et al., 1984) and there
are reports of exhumed seeds responding in the laboratory to
irradiations that established Pfr levels of
2% or less
(Taylorson, 1972). These findings and those of Baskin and
26
Baskin (1979) suggest that the VLFR mechanism might be
operative in buried seeds.
We have addressed this question by determining
fluence-response curves under laboratory and field
conditions for seeds of the arable weed Datura ferox that
had remained buried in the soil for some months. We chose
this species as test material because several aspects of its
germination ecology are already well known and seem to be
common to other annual dicots that invade rotation crops.
27
MATERIALS AND METHODS
The species
Datura ferox L.
(Solanaceae) is an aggressive annual
weed of summer crops in temperate and subtropical areas of
South America. Seeds are produced during late summer and
autumn and are dormant at maturity (Soriano et al., 1964).
In the field, germination of seeds kept on the soil surface
or buried at a constant depth is poor to nil. However,
flushes of seedling emergence take place during spring and
summer if, after several months of burial, the seeds are
returned near the soil surface (Soriano et al., 1971). Under
common agricultural practice, the main germination flushes
take place after events of soil cultivation (Ballare et al.,
1987, 1988).
Seed sources and burial procedures
Seeds of D. ferox were collected in the winter from
plants invading soybean fields in Lobos (Buenos Aires,
Argentina). Seeds of two different harvests were used: 1988
and 1989 (hereafter referred to as L88 and L89 batches,
respectively). The L88 batch was very heterogeneous
regarding the degree of seed maturity, as judged from the
28
color of the seed coats (Ballare et al., 1988), and was
divided into two pools, i.e. pale brown seeds (= light
coats) and dark brown seeds (= dark coats). After harvesting
the seeds were air dried and either stored in opaque glass
jars at constant 20°C, or buried to a depth of 7 cm in a
recently cultivated field at the Faculty of Agronomy
(University of Buenos Aires). This burial simulated the
effect of shallow cultivations which are normally used to
initiate fallow in the cropping areas where D. ferox is a
successful weed (Ballare et al., 1988). The physiological
status of the seeds at the time of burial is summarized in
Table 2.1. Plastic mesh bags each containing 50 seeds were
used in experiments where the seeds were exposed to light
after retrieval from the soil (i e.: laboratory experiments
and the simulated-cultivation experiment).
Supports of clear acrylic (Fig. 2.1A) were used in the
experiments where seeds were illuminated while buried in the
soil (in-situ irradiation experiment). These supports were
designed to guide light to the seeds. They consisted of a
sheet (20 x 20 x 4 cm thick) of transparent Paolini acrylic
(Paolini SA, Buenos Aires, Argentina) with a shelf attached
to one of the sides to hold the seeds. The seeds were placed
in a row on this shelf and were kept in place by a plastic
mesh. The face of the guide opposite to that used for sowing
was abraded to create a light-diffusing surface.
Except for
29
the portion that was left above ground (. 2.5 cm) and a
small window in front of the seeds, the acrylics were coated
with reflective polyester material. The light guides
carrying the seeds were buried in winter with their main
plane parallel to the predicted direction of the direct
solar rays at midday in late spring. The above ground
portion remained covered with a black cap until the time of
irradiations. Each guide was calibrated individually using
two LI 190SB quantum sensors (Li-Cor, NE, U.S.A.), one held
parallel to the light-entering, upper edge of the guide and
the other pointing at the seed window. Mean transmittance
was about 2%. Inspection of the guides after a period of
burial indicated that there was only minimal accumulation of
soil particles in the area of contact between the seeds and
the acrylic; therefore, the above figure was taken as a good
estimate of the actual transmittance during the experiment.
Light sources and irradiation treatments
R light was supplied by 40W/15K8 red fluorescent tubes
(Philips, Holland). FR was obtained by filtering light from
a 250-W incandescent lamp through a 5-cm thick water layer
and a RGN9 filter (Schott, FRG)
(Fig. 2.2A). To obtain the
different levels of Pfr, fluence rate was varied between 2.4
and 79.6 Amol 111-2
s-1
(R) or between 0.6 and 48.6 Amol m-2 s-1
(FR), and irradiation time between 2 seconds and 1 hour (R)
30
or between 5 seconds and 15 minutes (FR). Reciprocity was
found to hold for the longest irradiations used.
A dim green light was used for handling the seeds in
the dark room after retrieval from the soil. The source
provided <2 x 10-2 Amol m-2 s-1 between 400 and 550 nm at the
seed level. The seeds were exposed to this light for periods
shorter than one minute and this exposure had no effect on
germination.
Irradiations with sunlight were performed in two ways.
In the in situ irradiation experiment (Fig. 2.3A), sunlight
reached the seeds after passing through the light guide used
for burial. The average fluence rate at seed level was 59
Amol m2 s-1 (400-800 nm). Different fluences were obtained
by varying the irradiation time between 2 seconds and 2
hours. In the simulated-cultivation experiment (Fig. 2.3B),
seed bags were unearthed near midday using light proof
equipment. The bags were thoroughly shaken to eliminate
bound soil particles, transferred to a shutter box under dim
green light and then given a controlled exposure to
sunlight. Sunlight fluence rate was varied from 0.15 to
1,950 Amol m-2 s-1 (400-800 nm) by means of neutral density
filters. Exposure times were varied between 2 and 153
seconds. In the FR-reversion test the seeds were placed
under the FR source (Fig. 2.2A) within 2 minutes after
31
receiving 3 x 105 Amol m-2 of sunlight and were given 1.5 x
105 Amol m-2 in order to reduce the Pfr content to about 3%.
Relative humidity inside the irradiating cabinet was kept
near 100% to prevent seed desiccation. After irradiations
the bags were taken back to the dark room and placed
horizontally on a 3 cm thick layer of moistened soil that
was supported by means of a plastic mesh at the bottom of
opaque cylinders of PVC (10 cm high x 10 cm diameter). The
bags were then covered with a 7 cm thick soil layer and the
cylinders taken to the field and sunk into previously dug 10
cm deep holes. Except for the lack of light exposure, the
seeds used as dark controls were subjected to identical
manipulations. The number of germinated seeds was determined
two weeks after the irradiation treatment.
Measurements of seed coat transmittance and phytochrome
calculations
To determine the transmittance spectra, fragments of
the seed coats ('u 4 mm2) were bound with epoxy adhesive to a
perforated plate that was mounted on the light receiving
dome of the LI 1800 spectroradiometer (Li-Cor, NE, U.S.A.)
(Fig. 2.1B). The spectrum was scanned for light and dark
coats with the sample oriented perpendicular to the direct
sun rays. The coats were kept moist during measurements.
Reference spectra were obtained after removing the coats
32
from the mounting plate.
The amount of Pfr established by the different
irradiations was calculated as follows:
Pfr/P = 0 {1
exp -[ E
(al,x +cr,,x)Fx
] }
( 1)
where Fx = photon fluence at wavelength X after screening by
the seed coat (gmol m-2), al,), and cr2,x are the apparent
spectral molar conversion cross-sections for the transitions
Pr->Pfr and Pfr->Pr respectively (m2 gmol-1), P = total
phytochrome, and 0 = Pfr/P at photoequilibrium = E [(u,,x)Fx]
/ E
[(al,x +a2,x)Fx]. Eqn (1) was modified from Hartmann and
Cohnen-Unser (1972) to be used with polychromatic light
sources. To calculate the disappearance of Pfr in the FR-
reversion test (Figs. 2.3 and 2.4), eqn (1) was modified as
indicated by Cone and Kendrick (1985) to take account of
pre-existing Pfr in the seeds. The photoconversion cross-
sections used in these calculations were published by
Mancinelli (1988, Table 2.4) from the work of Kelly and
Lagarias (1985).
33
RESULTS AND DISCUSSION
Laboratory experiments
Seeds of Datura ferox that had been buried for two
months were retrieved in absolute darkness, placed on moist
cotton, and immediately given different fluences of R or FR
radiation (Fig. 2.2A). The pulses were designed to establish
different levels of Pfr in the seeds, which were calculated
analytically (Eqn 1) taking into account transmittance
spectra of the seed coats (Fig. 2.2B). After the light
treatments the seeds were transferred to darkness and given
an alternating-temperature regime (20 °C, 15 h; 30 °C, 9 h)
that was found to be favorable for the germination of D.
ferox (Soriano et al., 1964). The same treatments were given
to seeds of the same batch that had been maintained in dry
storage since the time of harvest.
Burial induced a dramatic increase in the sensitivity
of the seeds to light pulses forming very low % Pfr (Fig.
2.2C). When seeds that had been stored dry in the laboratory
for two months were tested we obtained the typical LFR
curve, where germination is negligible for Pfr values < 1%.
In seeds exhumed from soil, the levels of both, dark and
light-saturated germination was greater than for dry stored
seed, probably due to some overriding factor that influenced
34
germination independent of Pfr (Duke, 1978; Cone et al.,
1985). However the most remarkable observation was that most
of the exhumed seeds were induced to germinate when given R
or FR pulses that established less than 0.01% Pfr. There was
a good agreement between germination results produced by R
and FR when pulses established similar calculated %Pfr. The
shape of the germination: %-Pfr relationship, was strikingly
similar to those reported for laboratory treated seed
populations of other species. Two response regions were
found: one between 0.0001 and 0.01% Pfr, due to the
germination of seeds in the VLFR state, and the other
between 1 and 86% Pfr, due to the germination of seeds in
the LFR state (cf. Cone et al., 1985; Kronenberg and
Kendrick, 1986). These results clearly document, for the
first time, a massive, natural transition to the typical
VLFR state.
The mechanism whereby some seeds in the population
acquire the ability to respond to very low % Pfr is still
under debate (e.g. VanDerWoude, 1985; Cone et al., 1985). In
Datura it seems that low temperatures are not required for
sensitization. In fact (i) soil temperature at -7 cm did not
fall below 12°C during the burial period in the above
experiment, and (ii), we have obtained in the laboratory
seed populations displaying VLFR by keeping them for some
weeks in an atmosphere saturated with water vapour at
35
constant 25°C (A. L. Scopel, unpublished). This treatment is
known to produce a significant decrease in the level of
endogenous inhibitors (ABA among them) in seeds of D. ferox
(De Miguel, 1980). A similar loss of inhibitors may
conceivably take place in the field, while the seeds are
surrounded by a moist soil matrix, which might be important
for unmasking the VLFR mechanism in this species. Whatever
its physiological basis, the phenomenon of light
sensitization should be of considerable interest for
understanding the effects of soil cultivations on the
germination of weed seeds in arable lands. Soil disturbance
by tillage operations usually result in a flush of emergence
of weed seedlings (Chancellor, 1964; Roberts and Potter,
1980), and this is particularly notorious in areas infested
with D. ferox (Ballare et al., 1987, 1988). In many cases
light is a major factor in the germination response of the
seed bank; if steps are taken to exclude light, fewer
seedlings appear in the disturbed soil (Sauer and Struik,
1964; Wesson and Wareing, 1969; Hartmann and Nezadal, 1989).
A change in light sensitivity of the magnitude we have shown
in Fig. 2.2C is thus likely to influence the responses of
buried seeds to cultivation.
36
Field experiments
Extrapolating laboratory results to the field
situation requires caution. Relatively minor changes in the
conditions to which the exhumed seeds are exposed prior to
(Bouwmeester and Karssen, 1989) or during the germination
test (e.g. Baskin and Baskin, 1979; Bouwmeester and Karssen,
1989) can greatly affect the expression of the dormancy
status. Therefore, two additional experiments were
established to test for VLFR in the field while trying to
cause the least deviation from the natural condition. In one
experiment sunlight was piped from the soil surface to seeds
that had remained at -7 cm for 3 months after burial in
winter. This enabled us to study the light responses of
buried seeds without affecting other environmental factors
during illumination. At this depth, seeds of D. ferox do not
normally germinate in the field but can remain viable for at
least 20 months (Ballare et al., 1988). Seedling counts,
performed two weeks after the irradiation treatments
revealed that, under the conditions prevailing in the soil
in late spring (mean daily temperature > 25°C, mean daily
amplitude > 12°C), exposure to light was the only
requirement that had to be fulfilled in order to trigger
germination in about one-third of the seeds (Fig. 2.3A).
Moreover, even the lowest fluence delivered, which was
equivalent to 0.1 seconds of full sunlight, elicited some
37
germination. Additional experiments showed that light
germination was > 90% in seeds exhumed and tested under
standard laboratory conditions (not shown), indicating that
some overriding factor, independent of light and
temperature, reduced germination in the seeds maintained
at -7cm. The nature of this factor is unknown, but is
probably related to the conditions of poor aeration created
by soil compaction after three months.
In a second experiment we tested a wider range of
sunlight fluences following a protocol that more closely
mimicked the conditions experienced by seeds during soil
cultivation. Buried seeds were exhumed in darkness in late
spring, exposed to sunlight and buried again at the initial
depth. Retrieval and burial in the dark had no effect (cf.
Fig. 2.2C), but exposures to fluences equivalent to a few
milliseconds of full sunlight were sufficient to promote
germination in more than 50% of the seeds (Fig. 2.3B). FR
given immediately after a saturating pulse of sunlight
reduced germination, but only to the level induced by the
intermediate fluences. This result and calculations of the
amount of Pfr established by the various irradiations (Fig.
2.4A) indicate that m 80% of the light responding seeds were
in a VLFR state and required only between 0.0001 and 0.01%
Pfr to germinate under field conditions (Fig. 2.4B). Thus,
the experiment demonstrated that germination of D. ferox
38
seeds in response to soil disturbance was absolutely
dependent on light exposure and confirmed the enormous
sensitivity that was apparent in the laboratory study (Fig.
2.2C)
.
Ecological implications
To assess the ecological importance of the process of
sensitization, consideration should be given to the light
conditions encountered by seeds in arable soil. Provided
they are buried deeper than a few millimeters, the seeds
will be exposed to darkness (Woolley and Stoller, 1978;
Tester and Morris, 1987) for relatively long periods and,
eventually, to pulse-like irradiations with sunlight when
the soil is disturbed by cultivations. Because light
penetrates poorly through soil, only a small proportion of
the total seeds in the soil bank will have any chance of
perceiving further light after tillage is completed.
Moreover, the seeds left at or very near the soil surface
are exposed to higher risks of desiccation, photoinhibition
(Gorski and Gorska, 1979), and predation (Scopel et al.,
1988; Ballare et al., 1988). Therefore, the production of
germination flushes after soil cultivation events is likely
to be largely dependent on some seeds having the ability to
sense the light while the soil is being broken up by the
tillage implements. This reasoning is supported by recent
39
results of Hartmann and Nezadal (1989) who, using a farm-
scale version of the pioneer experiment of Sauer and Struik
(1964), found that weed seedling emergence in arable fields
can be strongly reduced if all soil disturbing operations
are performed during the night.
Taking mouldboard ploughing as an example, one can
obtain a rough estimate of the illumination times during
cultivation. At a normal working speed of 7 km 11-1, each
plough body lags . 0.25 seconds behind its immediate
predecessor. This means that the time elapsing between one
furrow is turned over and the moment in which it is
partially covered with soil pushed by the following
mouldboard will be of this order of magnitude. Exposures of
this kind are certainly too short to trigger a LFR, which
will require between 10 and 1000 times more photons (Figs.
2.3 and 2.4). In contrast, the % Pfr formed during such
exposure will be sufficient to give full germination in a
population of sensitized seeds (Figs. 2.3B and 2.4B).
40
CONCLUSIONS
Arable soils contain enormous quantities of viable
buried weed seeds and large flushes of germination typically
take place after the soil is disturbed by tillage
operations. Seeds of several weed species detect the
occurrence of soil cultivation events using light as an
environmental signal. In this paper we have shown that seeds
of the annual weed D. ferox undergo a large, . 10000-fold
increase in light sensitivity while they are buried in the
soil and that this phenomenon has a very marked effect on
the pattern of response to sunlight under field conditions.
According to our results, the induction of a highly
sensitive state is required for the detection by the seeds
of "split-second" exposures to sunlight. Because short
pulses of this nature are very likely to be the most
important light signals of soil cultivation, the natural
switch from the LFR to the VLFR state that we have
documented is probably essential for the perception of soil
disturbances by light-requiring weed seeds. Additional
experiments with actual disturbance regimes are warranted
and necessary to assess the general validity of this
hypothesis.
41
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dormancy. Nature 337: 645-646
Rethy R, Dedonder A, De Petter E, Van Wiemeersch L,
Fredericq H, De Greef J, Steyaert H, Stevens H (1987)
Biphasic fluence-response curves for phytochrome­
mediated Kalanchoe seed germination. Plant Physiology
83: 126-130
Roberts HA, Potter ME (1980) Emergence patterns of weed
seedlings in relation to cultivation and rainfall. Weed
Research 20: 377-386
Sauer J, Struik G (1964) A possible ecological relation
between soil disturbance, light-flash, and seed
germination. Ecology 45: 884-886
Scopel AL, Ballare CL, Ghersa CM (1988) Role of seed
reproduction in the population ecology of Sorghum
halepense in maize crops. Journal of Applied Ecology
25: 951-962
Smith H (1973) Light quality and germination: ecological
implications. In
W Heydecker, ed, Seed Ecology. The
Pennsylvania State University Press, University Park,
PA, pp 219-231
Soriano A, Sanchez RA, Eilberg BA (1964) Factors and
processes in the germination of Datura ferox L.
Canadian Journal of Botany 42: 1189-1203
45
Soriano A, Eilberg BA, Suero A (1971) Effects of burial and
changes in depth in the soil on the seeds of Datura
ferox. Weed Research 11: 196-199
Taylorson RB (1970) Changes in dormancy and viability of
weed seeds in soils. Weed Science 18: 265-269
Taylorson RB (1972) Phytochrome controlled changes in
dormancy and germination of buried weed seeds. Weed
Science 20: 417-422
Taylorson RB (1987) Environmental and chemical manipulation
of weed seed dormancy. Review of Weed Science 3: 135­
154
Taylorson RB, Dinola L (1989) Increased phytochrome
responsiveness and a high-temperature transition in
Barnyardgrass (Echinochloa crus-galli) seed dormancy.
Weed Science 37: 335-338
Tester M, Morris C (1987) The penetration of light through
soil. Plant, Cell and Environment 10: 281-286
VanderWoude WJ, Toole VK (1980) Studies of the mechanism of
enhancement of phytochrome-dependent lettuce seed
germination by prechilling. Plant Physiology 66: 220­
224
VanderWoude WJ (1985) A dimeric mechanism for the action of
phytochrome: evidence from photothermal interactions in
lettuce seed germination. Photochemistry and
Photobiology 42: 655-661
Wesson G, Wareing PF (1969) The role of light in the
46
germination of naturally occurring populations of
buried weed seeds. Journal of Experimental Botany 20:
402-413
Woolley JT, Stoller EW (1978) Light penetration and light-
induced seed germination in soil. Plant Physiology 61:
597-600
47
Table 2.1 Viability and germination of different seed batches of
Datura ferox at the time of burial (late winter)
Viability
Germination*
darkness
after R
Seed batch
L88, light coats
L88, dark coats
L89, dark coats
95
96
98
0
0
0
39
20
33
* Seeds were incubated under alternating temperatures. For
details, see Ballard et al.
(1988). Percent germination was
calculated based on the number of viable seeds.
48
A
lcm
B
1cm
Figure 2.1 (A) Light guides used to irradiate buried seeds
with sunlight in the field. (B) Mount used to hold the seed
coats during determinations of spectral transmittance.
Letter codes: a, acrylic light guide with abraded surface;
b, black cap; c, coat sample; d, teflon diffuser of the LI
1800 spectroradiometer; g, ground surface; m, mounting; n,
plastic net; 12, perforated plate; r, reflective coating; s,
seed; v, direct light vector.
49
VLFR
0
FR
A
R
>
<
LFR
>
80
1 FR
R
400
600
B
800
buried seeds
g 40
Ught
COCItS-....
8
8 60
10
dark
col3tsA
4
I
400
600
dry stored seeds
*
0
d
U'
a
800
WAVELENGTH (nm )
*5 3
10-2
161
io°
10t
102
Pfr IP(%)
Figure 2.2 (A) Relative spectral photon distribution of the
treatment light sources (LI 1800 spectroradiometer, Li-Cor,
NE, U.S.A.). (B) Transmittance spectra of the seed coats of
D.ferox. (C) Relationships between germination at 20°C /
Pfr established by
30°C in the laboratory and the
different fluences of R light (4, from 4.8 to 2.9 105 Amol
m-) or FR light (OS, from 3 and 4.4 104 Amol m-). The seeds
used as controls had been kept in dry storage at 20°C and
were soaked on moist cotton for 48 h at 25°C before
irradiations. Germination is expressed as percentage of the
number of viable seeds. Viability was > 95% (controls), and
> 90% (exhumed seeds). Crosses indicate the level of dark
germination; each datum point is the average of six
replicates of 50 seeds each; vertical bars represent ± 1 SE.
Only seeds with dark coats were used in these experiments
(batch L89).
50
(A) in situ irradiation
co ­
I,'
//...// /./
//....//
20
0
al
0
0
r-
102
103
106
los
z 100 -f113) simulated cultivation
0
P.
< .
8
z
80
1X
LU0
SL4 FR
-
f
60
if
A ,.1,
wNvor
20
0
4
I
o
10°
101
le
los
10'
102
101
SUNLIGHT (400.800nm) FLUENCE 4,m0(
0
10-`
10 2
102
10
10
101
102
FULL SUNLIGHT EQUIVALENT (s)
Figure 2.3 Fluence response curves for the induction of
germination by sunlight under field conditions. (A) Light
dark
light coats; 11
piped to buried seeds (batch L88;0
coats). Each datum point is the mean (± 1 SE) of 6
(B) Seeds exhumed in darkness,
replicates of 70 seeds each.
irradiated, and buried (batch L89, only dark seeds were
used). The diamond indicates germination of seeds irradiated
with FR after a saturating exposure to sunlight; each datum
point is the mean (± 1 SE) of six replicates of 50 seeds
each. Germination is expressed as percentage of the number
of viable seeds (viability was > 90t). As the experiments
were conducted with different seed batches and were not
concurrent, comparison between panels A and B is not
appropriate.
,
,
51
<
100
IRRADIATION TIME (s)
a0
VLFR
>
LFR
<
>
no,
B simulated cultivation
./
(8/
i--4,--4-----,
so
/46
mi
a
60
'74
a
O 40
A
/
in situ irradiation
O 20
Q
o
.
.
0
10
102
106
SUNUGHT FLUENCE ()mot m2)
id'
-3
10
.
2
10
-1
10
.
.
10°
101
.
e
Pfr/P ( '4 )
Figure 2.4 (A) Calculated Pfr/P ratios established by
different fluences of sunlight. (B) Relationship between
Pfr established by the various
germination and the
exposures to sunlight or sunlight followed by FR. Data from
Fig. 2.2.
52
CHAPTER 3
PHOTOSTIMULATION OF SEED GERMINATION DURING SOIL TILLAGE
Ana L. Scopel, Carlos L. Ballare and Steven R. Radosevich
Departments of Forest Science and Crop and Soil Science,
Oregon State University, Corvallis, OR 97331-7501, USA
(C.L.B., A.L.S., S.R.R); Departamento de Ecologia, Facultad
de Agronomia, University of Buenos Aires,
Aires, Argentina (C.L.B., A.L.S.).
New Phytologist, in press (1993)
(1417) Buenos
53
ABSTRACT
We conducted intensively replicated field experiments
in the Willamette Valley, Oregon, USA, in order to study the
role of light in triggering seed germination during soil
tillage. We found that the normal practice of cultivating
agricultural land during daytime can increase germination of
buried seed populations between 70 and 400
1,­
above the
levels recorded following nighttime cultivations.
Experimental reduction of the irradiance under the tillage
implements during daytime cultivation decreased the number
of dicotyledonous seedlings emerged, while strong artificial
illumination (> 300 Amol m-2 s-1; 400-800 nm) of the soil
surface under the implements during nighttime tillage
significantly increased seedling densities. These results
suggest that the enhancement of seed germination caused by
daytime tillage, compared with nighttime tillage, is due to
light that penetrates into the soil during the actual
disturbance. The detection by the seeds of this very short
exposure to sunlight requires a high photosensitivity, which
provides an adaptive "purpose" for the evolution of the
Very-Low-Fluence response mechanism in phytochrome­
controlled seed germination. Seedling emergence induced by
nighttime tillage was considerable in some experiments,
suggesting that light perceived by seeds after cultivation
or other microenvironmental factors affected by tillage may
54
be important in triggering germination in disturbed soil.
Key words: Germination ecology, Phytochrome, Tillage, Very­
Low-Fluence (VLF), Weed
55
INTRODUCTION
Soil tillage stimulates germination of buried seed
populations in arable land (Chancellor, 1964; Roberts and
Potter, 1980; Ballare et al., 1988). The possibility that
exposure of buried seeds to sunlight is the factor that
triggers germination in disturbed soil was suggested almost
three decades ago (Sauer and Struik, 1964; Wesson and
Wareing, 1969). This hypothesis is supported by the
following observations:
(i) Seeds of many weedy species or
ecotypes require light to germinate (Grime, 1979; Taylorson,
1987);
(ii) frequently, disturbance of soil samples in the
dark results in less germination than disturbance under
strong illumination (Sauer and Struik, 1964; Wesson and
Wareing, 1969);
(iii) in some weedy species, seed burial
induces a dramatic increase in light sensitivity, which has
been interpreted as a natural induction of the Very-Low-
Fluence (VLF) response mechanism (Scopel et al., 1991). The
VLF response is common in several processes mediated by the
photoreceptor phytochrome (Mandoli and Briggs, 1981;
reviewed by Kronenberg and Kendrick, 1986, and VanDerWoude,
1989), is triggered by light exposures that would form very
small amounts of the far-red- (FR-) absorbing form of
phytochrome (Pfr)
(i.e. between 10-4 and 10-2
Pfr [Cone et
al., 1985]), and may allow seeds to detect sub-millisecond­
exposures to sunlight when the soil is being disturbed by
56
tillage (Scopel et al., 1991).
In spite of this evidence, the quantitative importance
of light-induced germination in cultivated land remains
unclear. This is because:
(i) besides changing the light
conditions, soil tillage can alter many other aspects of the
soil environment that influence seed dormancy and
germination, and (ii), the responses of natural seed banks
to tillage cannot be adequately predicted by scaling-up from
physiological and single-factor studies (discussed by
Ballare et al., 1992). In a study that tested the light
requirements for germination of buried seeds, only one third
of a seed population of Datura ferox could be induced to
germinate in situ by saturating exposures to sunlight after
three months of burial. When the soil surrounding the seeds
was mechanically disturbed in a parallel experiment, the
proportion of buried seeds that germinated after exposure to
light increased by a factor of three, indicating that the
lack of light was unlikely to be the only factor limiting
germination in the soil (Scopel et al., 1991). A few field-
scale studies have compared weed seed germination induced by
daytime and nighttime cultivations with standard tillage
equipment, and the results have been mixed. Feltner (1967)
reported a 40 96 reduction in the emergence of Amaranthus
retroflexus in field plots cultivated at night. He
acknowledged frequent location x treatment interactions and
57
the variability in his data set was apparently too large to
allow any definitive conclusion (Woolley and Stoller, 1978).
Hartmann and Nezadal (1990) reported that a field strip
where tillage operations were carried out at night over a
six-year period had dramatically reduced weed abundances
compared with an adjacent strip cultivated near solar noon.
Jensen (1991) found that daytime cultivation resulted in
larger numbers of emerged seedlings in one out of two field
experiments. A reduction in seedling numbers with nighttime
tillage was apparent in a third field experiment, but
logistic problems encountered when applying one of the
treatments prevented statistical inference from the
resulting data. One problem is that replication intensity in
some of these studies could have been insufficient, given
the extreme spatial heterogeneity of soil seed banks in
agricultural fields (e.g. Goyeau and Fablet, 1982; Benoit et
al., 1989; Dessaint et al., 1991). Another problem is that
only the average effects of treatments across species have
been reported in some cases, and species vary in their light
requirements for germination (e.g. Grime, 1979; Bewley and
Black, 1982). An additional complication is that the light
requirement for germination of buried seeds may fluctuate
dramatically with time (Taylorson, 1972; Froud-Williams et
al., 1984; Baskin and Baskin, 1985; Scopel et al., 1991),
and may be affected by the light environment experienced by
the seeds before burial (Fenner, 1980).
58
Overall, the critical question that has not been
directly addressed in field experiments is to what extent
light signals perceived by the seeds when the soil is being
actually cultivated contribute to germination induction.
Light penetrates poorly through soil, but penetration may be
increased by cracks and air spaces among aggregates in well
structured agricultural soil (see Bliss and Smith, 1985;
Tester and Morris, 1987; Mandoli et al., 1990). Thus, a
potentially large number of seeds left near the soil surface
by cultivation might receive light stimuli after tillage,
and eventually contribute to the flux of germination. Recent
results with Datura ferox show that, at least for seed
batches that display FR-reversible photoresponses (i.e. the
classical, low-fluence response (LF, Kronenberg and
Kendrick, 19861), light promotion of seed germination is
mediated by a phytochrome pool that is highly stable in the
Pfr form (presumably phytochrome B)
(Casal et al., 1991).
This observation suggests that even very weak light received
by seeds left under a few millimeters of soil might induce
germination, because the light signal could be integrated
(as Pfr) over several photoperiods (Ballar6 et al., 1992).
This paper presents results of large-scale field
experiments designed to specifically test the role of light
received by seeds during soil disturbance in the promotion
of germination caused by tillage.
59
Abbreviations: FR, far red (700-800 nm); LF, low fluence;
Pfr, FR-absorbing form of phytochrome; VLF, very low
fluence.
60
MATERIALS AND METHODS
Study site and general experimental conditions
The experiments were conducted in the field at the
Vegetable Research Farm (Dept. of Horticulture, Oregon State
University) located on well drained silty clay loam soils of
the Chehalis series in the Willamette Valley, near
Corvallis, OR, USA. The annual precipitation is 960 mm and
is concentrated in the autumn and winter months. The farm is
divided into several experimental fields on the basis of
topographic position, soil characteristics, and cultural
history. All fields used in these studies (typically between
0.1 and 0.25 ha) had been planted with vegetable crops
during the last 40 years and presented a rich flora of weedy
annual species. The most conspicuous summer-annuals found in
the experimental plots were Amaranthus retroflexus L.
(redroot pigweed) and Solanum nigrum L. and S. sarrachoides
Sendtner (black and hairy nightshade, respectively). The
predominant species after the late-summer / early-autumn
tillage were Poa annua L.
(annual bluegrass), Sonchus spp.
(sowthistles), Lamium purpureum L.
(red deadnettle),
Cerastium vulgatum L. (mousear chickweed), Stellaria media
(L.) Vill. (common chickweed), Anthemis cotula L.
fennel), and Veronica spp. (speedwells).
(dog
61
For each experiment, one or two apparently uniform
experimental fields were selected in the farm, and within
each field, treatments were arranged in randomized complete
blocks. Between 6 and 26 replicate blocks were used in each
experiment depending on the size of the available fields and
the number of treatments (see Table 3.1 and also legends for
Figs. 3.1
3.4). Each experimental plot (i.e. block x
treatment combination) was 2 m by 18 m and was oriented with
its long axis perpendicular to the long axis of the field.
Typically, the integrated area of the plots occupied between
60 and 80 % of the experimental field area. All experimental
cultivations were carried out with a dual-action disc harrow
or with a mould-board plough followed by a dual-action disc
harrow during summer and early autumn. In each experiment,
all tillage treatments were applied within a time period of
less than 12 h. The number of harrow passes was adjusted in
each experiment to obtain a distribution of soil-aggregate
sizes comparable with that of a typical seed bed. In each
individual plot, all passes were completed in <15 min.
Cultivations were performed by the authors and care was
taken to ensure that the working speed and the settings on
the tillage equipment were the same for all cultivation
treatments. Daytime cultivations were performed around noon
under unobstructed sunlight. Nighttime cultivations started
more than 3 h after the official sunset. The only light that
could be seen during control nighttime cultivations came
62
from the stars and the lighting system of the distant town.
The nominal working speed was 7 km 11-1. Ploughing depth was
-18 cm. The mould-board plough had two 14-inch (35.6-cm)
blades. The disc harrow was of the dual-action type, had a
working width of 163 cm and consisted of two 15-inch (38.1
cm) disk packs followed by a 17.5-inch (43.8-cm) roller. The
clearance between disc packs varied between 27 cm (center)
to 47 cm (outer border of the harrow). Specific details on
the tillage sequences and experimental manipulations of the
light environment are given below.
1991 Experiments
In a first series of experiments, performed in early
summer of 1991, we studied the effects of daytime and
nighttime cultivation on seedling emergence. The previous
crop in the field was squash; a superficial cultivation was
applied one month before the experimental tillage using a
rotary harrow. The experimental cultivations were carried
out in July 1991 with a mould-board plough followed by two
passes with a disc harrow. The field was sprinkle irrigated
within 48 h before cultivation; no irrigation was applied
after tillage.
In another experiment (late summer) we tested the
effect of artificial weak illumination (- 30 Amol 111-2
s-1)
63
during the night. This experiment was carried out in a field
that had been planted with squash and field beans in the
previous summer. A superficial cultivation was applied one
month before the experimental tillage using a rotary harrow.
The experimental cultivations were carried out in August
1991 with a mould-board plough followed by two passes with a
disc harrow (timing and details as described above). In
order to provide additional illumination during tillage time
only, a light bank was mounted on the tillage equipment and
switched on at the beginning and off at the end of each
tillage pass. The light bank consisted of ten 12-volt
automotive headlights equipped with 55-W halogen bulbs
(Durimex H3). The irradiance (400
800 nm) at soil level
between the disc packs was 31 ± 1 Amol m-2 s-1 (n = 60) and
was estimated from actual readings for the visible wave­
band, taken with a Li-Cor (LI-185A) radiometer, and the
spectral distribution of the light source (determined with a
LI-1800 spectroradiometer). The plots were irrigated 3 wk
after the experimental cultivations.
In the autumn of 1991 we tested the effect of covering
the tillage implements during cultivation under full
sunlight. The experimental cultivations were carried out in
October 1991 with two passes of a disc harrow (details as
above) on the stubble of a bean crop. To obtain the "cover"
treatment, the top and four sides of the harrow were covered
64
with sheets of corrugated cardboard and with a heavy, opaque
tarpaulin. These covers filtered out all direct sunlight.
Some diffuse light penetrated into the working zone through
small and transient openings formed between the covers and
the soil surface as the equipment advanced over the
irregular ground. No irrigation was applied before or after
cultivation.
1992 Experiments
During 1992, large numbers of replicate plots were
cultivated during the summer and autumn at (i) midnight with
or without strong (>300 Amol 111-2 s-1) artificial
illumination, and (ii) midday with or without the implements
covered to reduce the irradiance at ground level. For
practical reasons, all the experimental cultivations were
performed with a disc-harrow and the observations were
concentrated on dicotyledonous species. Cultivations were
carried out in June or September with three passes of the
disc harrow (details as above) on fields that had been
ploughed at night in late spring (summer tillage) or
ploughed at night and cultivated at least once during the
summer (autumn tillage). The experimental manipulations of
the light conditions consisted of reducing the irradiance
during daytime cultivation by covering the disc harrow
(details as in 1991) or providing strong additional
65
illumination during the night with lamps mounted on the
tillage equipment. The light bank consisted of 10 automotive
headlights equipped with 55-W halogen bulbs (Durimex H-3)
and four 110-volt, 500-W quartz-halogen floodlight units
(Regent Lighting Co.) powered by a portable AC generator
mounted on the harrow. The irradiance (400
800 nm) at soil
level between the disc packs was 317 ± 14 Amol m-2 s-1
(n =
32), which was about 50 % of the irradiance measured between
the disc packs of the same harrow under clear-sky conditions
[617 ± 54 Amol m-2 s-1
(n = 39)].
Sampling and statistics
Between eight and 20 quadrats (0.40 or 0.05 m2) were
used to sub-sample each plot depending on seedling density.
Sub-samples were pooled to obtain a block average, which was
used in all subsequent analyses (Hurlbert, 1985). For each
block, the number of seedlings emerged in a given
cultivation treatment was divided by the corresponding
control. In the figures, we present the calculated ratios
(treatment / control) averaged across the N blocks and their
standard errors. ANOVA was performed for each experiment and
the error mean square was used to calculte the Fisher's
Protected Least Significant Difference (FPLSD)
(Stafford,
1991). This multiple comparison test was used to determine
significant differences between the mean ratios of the
66
individual treatments and their corresponding controls (i.e.
ratio = 1). The ANOVA and FPLSD tests for each experiment
are included in Appendix 1 (pp. 140-151).
67
RESULTS
1991 Experiments
Compared with nighttime tillage, cultivation with a
mould-board plough and a disc harrow near solar noon in
early summer had a dramatic impact promoting seedling
emergence in a field dominated by Amaranthus retroflexus and
Solanum spp.
(Fig. 3.1; Appendix 1, p. 140). In another
field, which was cultivated in late summer with the same
implements, nighttime tillage was followed by a considerable
flux of seedling emergence; yet, daytime cultivation caused
a two-fold increase in the germination of winter-annual
dicotyledonous and grass species relative to that nighttime
tillage control (Fig. 3.2; Appendix 1, p. 142). The
additional weak illumination (<30 Amol m2 s-1) during
nighttime tillage had no effect on seedling emergence
compared with the dark control (Fig. 3.2; Appendix 1, p.
142). A similar pattern of response was observed in a late-
germinating cohort of the summer-annual species A.
retroflexus and Solanum spp.
(not shown).
As a more rigorous test of the role of light perceived
by seeds during tillage in the induction of germination, we
cultivated the soil at midday with the top and sides of the
tillage implements covered to prevent sunlight from reaching
68
the ground surface. This treatment was intended to interfere
only with seeds that received sunlight in the form of a
flash exposure while the surrounding soil was being
disturbed by the tillage implement; after cultivation the
soil surface was left exposed to direct sunlight. Covering
the disk harrow during an autumn tillage caused a 30
96
reduction in the number of dicotyledonous (winter-annuals)
seedlings emerged compared with the uncovered control. The
emergence of grass species was not affected by the covering
treatment (Fig. 3.3; Appendix 1; p. 145). This experiment
demonstrated that sunlight sensed by dicotyledonous seeds
during tillage can promote their germination in the field,
and was in apparent conflict with the results from the
artificial-lighting experiments described above.
Several factors could have contributed to this
apparent discrepancy, including: (i) differences between
experiments in the species composition of the soil seed
bank,
(ii) differences in the dormancy status of the seed
bank,
(iii) unknown endogenous or environmental conditions
during the night that prevent photostimulation, and (iv),
insufficient irradiance in the artificial-lighting
experiment. The first possibility is weakened by the
observation that the species lists for the seedling cohorts
that followed the late summer (Fig. 3.2) and autumn (Fig.
3.3) tillage have several species in common under the group
69
of winter-annual dicots. The fourth possibility was
addressed theoretically by roughly estimating the amount of
Pfr that could have been formed in the seeds after an
exposure to the light provided by our light bank. These
calculations were performed using the equations and
attenuation properties of seed coats described by Scopel et
al.
(1991). Assuming an exposure time for individual seeds
of 0.14 to 0.25 s (the times required by the harrow to cover
the distances between disk packs at 7 km 11-1 [see Materials
and Methods]), and an irradiance of 31 Amol 111-2 s-1, we
estimated the Pfr level to be between 3 x 10-3 and 6 x 10-3
96. This Pfr level should induce germination of the most
sensitive fraction of a seed population responding in the
VLF range (i.e.: 10-4 to 10-2 li-Pfr, Cone et al., 1985, see
Introduction). However, the actual amounts of Pfr
established in the seeds during cultivation were almost
certainly lower than this estimate, due to attenuation of
the actinic light by dust and soil particles adhered to the
seeds.
1992 Experiments
Of course, the above calculations only provide a
coarse approximation to the light conditions sensed by
phytochrome in individual seeds during tillage. Therefore,
in order to test the irradiance hypothesis more directly, we
70
increased the output of the implement-mounted light source
by an order of magnitude in the experiments of 1992,
obtaining an average surface irradiance that was only 50 %
lower than that measured between the disc packs under clear-
sky conditions. Using the same approach explained above, the
Pfr levels established on individual seeds by direct
exposure to this light were estimated to be between 3.7 x
10-2 and 6.6 x 10-2 %. These Pfr levels are four to seven
times greater than the required to saturate a VLF-response,
but still two orders of magnitude lower than what would be
necessary to trigger a typical FR-reversible, LF response
(Cone et al., 1985; Scopel et al., 1991).
As in 1991, midday cultivation in the summer and
autumn resulted in increased emergence of dicotyledonous
seedlings, compared with the midnight tillage control (Fig.
3.4; Appendix 1, p. 148). Covering the tillage implement
during daytime cultivation reduced seedling emergence to
levels that were similar to those obtained after nighttime
tillage (Fig. 3.4; Appendix 1, p. 148). Irradiation of the
soil under the implements with - 300 Amol m-2 s-1 during
midnight cultivation increased the number of seedlings
emerged by about 50 11, compared with the dark control (Fig.
3.4; Appendix 1, p. 148). The patterns of response to the
experimental manipulations of the light environment observed
in the summer and autumn experiments were very similar.
71
However, it has to be pointed out that the absolute
germination level in the plots cultivated during nighttime
was large in the experiment performed at the beginning of
the rainy season (Fig. 3.4, autumn; see also Fig. 3.2).
72
DISCUSSION
The experiments reported in this paper show that (i)
nighttime cultivation (Figs. 3.1, 3.2, and 3.4) and
cultivation with covered tillage implements (Figs. 3.3, and
3.4) result in reduced emergence of dicotyledonous seedlings
compared with cultivation under full sunlight, and (ii) the
reductions in seedling emergence caused by the two
treatments (i.e. nighttime tillage or tillage under an
opaque cover) are quantitatively similar (Fig. 3.4). The
simplest interpretation of this result is that most of the
promotion of germination caused by daytime tillage (compared
with nighttime tillage) is caused by very short exposures of
the seed to sunlight when the soil matrix is mechanically
disturbed. In agreement with this hypothesis, we found that
irradiation of the soil under the implements with strong
light (- 300 Amol m2 s-1) during midnight cultivation
significantly increased the number of seedlings emerged
compared with the dark control (Fig. 3.4). This observation
is also consistent with the hypothesis that a VLF-sensitive
mechanism is responsible for the detection of the brief
light exposure because, according to our calculations (see
above), the artificial light treatment would have formed <
10-1 % of Pfr in the exposed seeds, and Pfr levels > 3
'4,­
required in order to effect a LF response (Cone et al.,
1985)
.
are
73
An important observation requiring further discussion
is that, although the results of this series of experiments
(Figs. 3.1, 3.3, and 3.4) point to the importance of light
perceived during tillage to the promotion of germination,
the absolute germination level in the plots cultivated
during nighttime was considerable in some cases (Figs. 3.2
and 3.4, autumn). A relatively large background germination
after nighttime tillage was also observed by Jensen (1991)
working in Denmark. We have established that, in our
experiments and at least for the dicotyledonous species,
most of this germination is indeed triggered by soil tillage
(i.e. seedling emergence is negligible in unperturbed soil;
data not shown). Our data do not allow us to distinguish
whether these seedlings originate (i) from seeds that are
not photoblastic and are triggered to germinate by other
effects of tillage on the soil microenvironment (e.g.
altered temperature), or, alternatively,
(ii) from seeds
that receive and respond to light during the days that
follow the tillage operation. As already mentioned,
photostimulation of seed germination (by LFs) in some
species involves the action of a phytochrome pool that is
highly stable as Pfr (presumably phytochrome B)
(Casal et
al., 1991). It is not known if the VLF response is also
mediated by a stable phytochrome, but clearly the
possibility exists that hydrated seeds are able to integrate
light signals (LF, and possibly VLF as well) received over a
74
relatively long period of time. In this regard we have
noticed that, during the rainy autumn in the Willamette
Valley, the surface of the soil may remain near field
capacity for several days. Therefore, the favorable moisture
conditions during the autumn might support germination of
seeds left by cultivation in microsites exposed to light
close to the soil surface. Following this line of thought,
one should expect the largest differences between daytime
and nighttime cultivations in conditions that favor rapid
desiccation of the upper soil profile. Interestingly, the
largest difference between the two treatments was observed
in an experiment carried out in mid summer (Fig. 3.1). On
the other hand, and to the extent that tillages modify the
vertical distribution of seed populations in the soil
profile (e.g. Van Esso et al., 1986; Cousens and Moss, 1990,
and references therein), they would also modify the thermal
environment sensed by the seeds. Thermal cues are important
for germination in many species (Grime, 1979), including
weeds of arable land (Benech Arnold et al., 1988). The
effect of tillage on the thermal environment, rather than
exposure to light, may be the critical factor triggering
germination in seeds that respond to nighttime tillage.
Another important point is that the promotion of
germination caused by daytime tillage (compared with
nighttime tillage), was generally larger when the
75
cultivations involved the use of a mould-board plough (Figs.
3.1 and 3.2) than when cultivations were carried out with a
disk harrow only (Fig. 3.4). A likely explanation may lie on
the fact that the disc harrow causes much less soil
inversion than the mould-board plough, which is known to be
very effective at moving seeds between depth layers during
soil cultivation (Van Esso et al., 1986; Cousens and Moss,
1990, and references therein). The large difference
(particularly in the summer) between daytime and nighttime
cultivations with a mould-board plough (Fig. 3.1) may be due
to the germination of seeds that were left in soil layers
protected from desiccation after being photostimulated by a
VLF pulse during tillage.
The response of the grasses to manipulations of the
light environment during tillage were not consistent among
experiments (Figs. 3.2 and 3.3), in spite of the fact that a
single species (Poa annua) was the dominant component of the
grass flora in all cases. A clear difference between
treatments was only seen in the summer experiment of 1991
(Fig. 3.2), where daytime cultivation promoted germination
by a factor of 2.4 compared with nighttime cultivation.
Covering the tillage implements to reduce the irradiance in
the working zone (Fig. 3.3), or cultivating during nighttime
(not shown) had no effect on grass seedling emergence in the
autumn experiments. A possible explanation of these results
76
is that the light requirement fluctuates with time in the
seed bank. Changes in light sensitivity during burial have
been documented in many studies (e.g. Taylorson, 1972;
Froud-Williams et al., 1984; Baskin and Baskin, 1985; Scopel
et al., 1991). In studies with Poa annua in Great Britain
(Froud-Williams et al., 1984) it was found that the level of
dark germination of exhumed seeds increased dramatically
after six months of burial. In Datura ferox, the VLF
response mechanism is rapidly induced in the buried seeds
(Scopel et al., 1991), but at least under certain
experimental conditions (optimum alternating-temperature
regime), the light requirement may eventually disappear with
time (Scopel et al., 1991 and unpublished results). A
second, alternative explanation for the variability observed
with the grasses in the present experiments is that the
expression of the light requirement is affected by other
environmental conditions experienced by the seeds after
tillage, which may fluctuate on a seasonal basis.
Bouwmeester and Karssen (1989) have demonstrated that
factors such as addition of nitrate to the incubation medium
affected the response of exhumed Chenopodium album and
Spergula arvensis seeds to light, and Baskin and Baskin
(1985) have shown that the responses of exhumed Panicum
capillare seeds to light depended on the temperature regime
to which the seeds were exposed after the light treatment.
Carefully planned experiments will be required to
77
distinguish between these possibilities (i.e. variation in
light sensitivity or variation in the environmental
conditions that affect the expression of light sensitivity),
and to assess the extent to which the response of a given
species group to the tillage light environment changes over
time.
78
CONCLUSIONS
We conclude from these experiments, which involved a
very high degree of spatial replication, that the normal
practice of cultivating agricultural fields during daytime
enhances germination of dicotyledonous weed seeds, compared
with nighttime cultivation (Figs. 3.1, 3.2, and 3.4). Under
our environmental conditions, the mean enhancement factor
for dicotyledonous species averaged over all blocks and
experiments was 2.2 (S.E. = 0.2, N [number of blocks]
= 70).
The range of response for individual blocks was 0.2 to 10.5.
This emphasizes the critical need of intensive spatial
replication in studies of environmental control of
germination with natural seed banks. Our experiments further
indicate that the germination enhancement caused by daytime
tillage in dicotyledonous species is due to light that
penetrates into the soil during the actual disturbance (Fig.
3.4). The detection by phytochrome of this very short
exposure to sunlight requires a very high sensitivity, and
it is proposed that the photodetection of soil disturbances
is one of the major adaptive "goals" that favored the
evolution of germination responses through the VLF
mechanism. According to our results, reducing the irradiance
at the ground surface during tillage (i.e. by using
nighttime cultivation or light-shielded equipment) may
contribute to reduce weed seedling emergence in agricultural
79
land and may be an useful complementary tool in weed control
programes.
Acknowledgments
Supported by the U.S. Department of Agriculture, the
Consejo Nacional de Investigaciones Cientificas y Tecnicas
(Argentina), and the Dept. of Forest Science (Oregon State
University, USA). We thank L. C. Burrill for helping with
species identification, R. Hopson for practical assistance,
and C. M. Ghersa and an anonymous reviewer for comments and
suggestions on the manuscript.
80
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Ballare CL, Scopel AL, Sanchez RA, Radosevich SR (1992)
Photomorphogenic processes in the agricultural
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788
Baskin JM, Baskin CC (1985) Seasonal changes in the
germination responses of buried witchgrass (Panicum
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Benech Arnold RA, Ghersa CM, Sanchez RA, Garcia Fernandez AE
(1988) The role of fluctuating temperatures in the
germination and establishment of Sorghum halepense (L.)
Pers. Regulation of germination under leaf canopies.
Functional Ecology 2: 311-318
Benoit DL, Henkel NC, Cavers, PB (1989) Factors influencing
the precision of soil seed bank estimates. Canadian
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Bewley JD, Black M (1982) Physiology and Biochemistry of
Seeds in Relation to Germination, Vol. 2: Viability,
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Bliss D, Smith H (1985) Penetration of light into soil and
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Cell and Environment 8: 475-483
Bouwmeester HG, Karssen CM (1989) Environmental factors
influencing the expression of dormancy patterns in weed
seeds. Annals of Botany 63: 113-120
Casal JJ, Sanchez RA, Di Benedetto AH, De Miguel LC (1991)
Light promotion of seed germination in Datura ferox is
mediated by a highly stable pool of phytochrome.
Photochemistry and Photobiology 53: 249-254
Chancellor RJ (1964) Emergence of weed seedlings in the
field and the effects of different frequencies of
cultivation. Proceedings of the 7th British Weed
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response curves for light induced germination of
Arabidopsis thaliana seeds. Plant, Cell and Environment
8: 605-612
Cousens R, Moss SR (1990) A model of the effects of
cultivation on the vertical distribution of weed seeds
within the soil. Weed Research 30: 61-70
Dessaint F, Chadoeuf R, Barralis G (1991) Spatial pattern
analysis of weed seeds in the cultivated soil seed
bank. Journal od Applied Ecology 28: 721-730
Feltner KC (1967) Light requirements for weed seed
germination. Proceedings of the North Central Weed
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Fenner M.
(1980) The induction of a light requirement in
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Bidens pilosa seeds by leaf canopy shade. New
Phytologist 84: 103-106
Froud-Williams RJ, Drennan DSH, Chancellor RJ (1984) The
influence of burial and dry-storage upon cyclic changes
in dormancy, germination and response to light in seeds
of various arable weeds. New Phytologist 96: 473-481
Goyeau H, Fablet G (1982) Etude du stock the semences de
mauvaises herbs dans le sol: le probleme de
l'echantillonnage. Agronomie 2: 545-552
Grime JP (1979) Plant Strategies and Vegetation Processes.
John Wiley and Sons, Chichester
Haetmann KM, Nezadal W (1990) Photocontrol of weeds without
herbicides. Naturwissenschaften 77: 158-163
Hurlbert SH (1985) Pseudoreplication and the design of
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Jensen PK (1991) Udnyttelse of ukrudtsfro's behov for
lysinduktion. 8. Danske Plantevmrnskonference, Ukrudt.
Tidsskrift for Planteavls Specialserie, Beretning nr.
S2110, Statens Planteavlsforsog. Lyngby, Denmark, pp
215-230
Kronenberg GHM, Kendrick RE (1986) The physiology of action.
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in Plants. Martinus Nijhoff, Dordrecht, pp 99-114
Mandoli DF, Briggs WR (1981) Phytochrome control of two low­
irradiance responses in etiolated oat seedlings. Plant
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Some spectral properties of several soil types:
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Roberts HA, Potter ME (1980) Emergence patterns of weed
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Sauer J, Struik G.
(1964) A possible ecological relation
between soil disturbance, light-flash, and seed
germination. Ecology 45: 884-886
Scopel AL, Ballara CL, Sanchez RA (1991) Induction of
extreme light sensitivity in buried weed seeds and its
role in the perception of soil cultivations. Plant
Cell, and Environment 14: 501-508
Stafford SG (1991) Natural Resources Data Analysis Course
Guide. Department of Forest Science, Oregon State
University, Corvallis, OR
Taylorson RB (1972) Phytochrome controlled changes in
dormancy and germination of buried weed seeds. Weed
Science 20: 417-422
Taylorson RB (1987) Environmental and chemical manipulation
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154
Tester M, Morris C (1987) The penetration of light through
soil. Plant, Cell and Environment 10: 281-286
84
VanDerWoude WJ (1989) Phytochrome and sensitization in
germination control. In RB Taylorson, ed, Recent
Advances in the Development and Germination of Seeds.
Plenum Press, New York, pp 181-189
Van Esso ML, Ghersa CM, Soriano A (1986) Cultivation effects
on the dynamics of a Johnson grass seed population in
the soil profile. Soil and Tillage Research 6: 325-335
Wesson G, Wareing PF (1969) The role of light in the
germination of naturally occurring populations of
buried weed seeds. Journal of Experimental Botany 2:
402-413
Woolley JT, Stoller EW (1978) Light penetration and light-
induced seed germination in soil. Plant Physiology 61:
597-600
85
Table 3.1 Cultivation experiments carried out during 1991
and 1992
BLOCKS
YEAR
SEASON
TREATMENTS*
(N°)
Daytime
Cover
Nightime
Nightime
Illumination@
1991
SUMMER
Early
10
(July)
Weak
6
X
October
12
X
X
June
22
X
X
X
Strong
26
X
X
X
Strong
Late
X
(August)
AUTUMN
1992
SUMMER
AUTUMN September
X indicates treatment performed in that experiment.
@ Weak = 31 Amol m-2 s-1; Strong > 300 Amol m-2 s-1.
86
41
5t1
6.0 ­
O
E 5.0 ­
0
O .E 4.0­
Z
arg)
c
3.0 ­
.5
C/) 0
2.0­
E to
cv
Amaranthus
Solanum
retroflexus
spp.
Species
Figure 3.1 Effect of daytime cultivation in early summer
(1991) on seedling emergence compared with the nighttime (no
light) control. Absolute densities after nighttime
cultivations are indicated at the top in plants m2. Thin
bars indicate ± 1 S.E.; N (number of blocks) = 10. The weedy
flora (surveyed one month after cultivation) was dominated
almost exclusively by Amaranthus retroflexus, Solanum nigrum
Statistical analysis in Appendix 1.
sarrachoides
and S.
.
87
2.5
166131
2.0
Winterannual dicots
1.5
1.0
0.5
0
3.0
2391 71
Grasses
2.5
2.0
1.5
1.0
-
0.5
Daytime
Nighttime +
low light
Treatment
Figure 3.2 Effect of daytime cultivation in late summer
(1991) on seedling emergence compared with the nighttime (no
light) control. Also shown is the effect of weak artificial
illumination (31 Amol m2 s-1) during the night tillage.
Absolute densities after nighttime cultivations are
indicated at the top of each panel in plants m-2 (counts
made 5 wk after the experimental cultivations). Thin bars
indicate ± 1 S.E.; N (number of blocks) = 6. The most
conspicuous winter annual dicots were: Lamium purpureum,
Sonchus spp., Cerastium vulgatum, Veronica spp. and
Stellaria media. The most abundant grass was Poa annua.
Statistical analysis in Appendix 1.
88
284± 26
Winter
Annual Dicots
2381-20
Grasses
Species Group
Figure 3.3 Effect of covering the tillage implements during
daytime cultivation in the autumn (1991) on seedling
emergence compared with the full sunlight (no cover)
control. Absolute densities in the control plots are
indicated at the top in plants m' (seedling counts
performed 3 wk after the experimental cultivations). Thin
bars indicate ± 1 S.E.; N (number of blocks) = 12. The
predominant species among the winter annual dicots were:
Lamium purpureum, Cerastium vulgatum, Veronica spp.,
Stellaria media. Less abundant were Anthemis cotula and
Calandrinia ciliata var. menziesii. The most conspicuous
grass was Poa annua. Statistical analysis in Appendix 1.
89
Autumn
2.0
7)
2.0
O
1.0
1.8
E
1.6
1.6
0
1.4
1.4
1.2
1.2
1.0
1.0
0.8
0.8
6
0.6
0.6
c
0.4
0.4
0.2
0.2
366t 54
Summer
33t4
O
o
711
Tu.
0
0
Daytime
Daytime
w/cover
Nighttime
+ light
Daytime
Daytime
w/cover
Nighttime
+ light
Treatment
Figure 3.4 Effect of manipulating the light conditions
during cultivation on the emergence of dicotyledonous
seedlings compared with the nighttime (no light) control.
Absolute densities after nighttime cultivations are
indicated at the top of each panel in plants m-2 (seedling
counts performed 3 wk after the experimental cultivations).
Thin bars indicate ± 1 S.E.; N (number of blocks) = 22
(summer 1992, left panel) or 26 (autumn 1992, right panel).
The species composition of the plant communities established
after cultivation was as indicated in the legends for Figs.
3.1 (summer tillage) or 3.3 (autumn tillage). Statistical
analysis in Appendix 1.
90
CHAPTER 4
ABSCISIC ACID LEVELS AND THE INDUCTION OF THE VERY-LOW­
FLUENCE RESPONSE IN PHYTOCHROME CONTROLLED SEED GERMINATION
ANA L. SCOPEL, CARLOS L. BALLARE, ANITA N. AZARENKO, and
STEVEN R. RADOSEVICH
Departments of Forest Science and Crop and Soil Science,
Oregon State University, Corvallis, OR 97331-7501, USA
(C.L.B., A.L.S., S.R.R); Departamento de Ecologia, Facultad
de Agronomia, University of Buenos Aires,
(1417) Buenos
Aires, Argentina (C.L.B., A.L.S.); and Department of
Horticulture, Oregon State University, Corvallis, Oregon
97331, USA (A. N. A.)
91
ABSTRACT
A short period of burial in the soil induces a
dramatic increase in light sensitivity in seeds of the
annual plant Datura ferox L., which has been interpreted as
a natural transition to the very-low-fluence (VLF) mode of
phytochrome action (A.L. Scopel, C.L. Ballare, R.A. Sanchez
[1991] Plant Cell Environ 14: 501-508). In the work reported
in this paper, the connection between induction of
sensitivity to VLFs and endogenous abscisic acid (ABA)
levels was investigated by measuring the time courses of ABA
loss and induction of VLF-responsiveness during burial and
by investigating the effects of exogenous ABA on light
sensitivity. Our results indicate that (a) endogenous ABA
levels decrease during seed burial preceding or
concomitantly with the natural induction of the VLF
germination response;
(b) the total quantity of endogenous
ABA in buried seeds and the levels of R-induced or dark
germination are not obviously correlated;
(c) incubation of
the seeds in an atmosphere saturated with water vapor at
25°C reduces endogenous ABA levels and renders the seeds
sensitive to VLFs;
(d) addition of ABA to the seed
incubation medium has a greater negative impact on the VLF
response than on the response to saturating red light
pulses;
(e) the quotient between far-red- (VLF-) and red-
induced germination tends to decrese with endogenous ABA in
92
seed samples that differ in ABA content as a consequence of
natural ABA loss or experimental ABA addition. These results
are consistent with the hypothesis that the reduction in
endogenous ABA levels during seed burial plays a role in
switching the seeds from the low fluence to the VLF mode of
phytochrome action, and suggest that the mechanism whereby
ABA reduces the responsivity to VLFs is, at least to some
extent, independent of the inhibitory effect(s) of ABA on
seed germination.
Abbreviations: FR, far-red light; LF, low fluence; R, red
light; VLF, very low fluence; WSA, water-vapor saturated
atmosphere; X and Y, hypothetical reactants in the
phytochrome signal transduction chain.
93
INTRODUCTION
Light, acting through phytochrome, is an important
environmental cue for seed germination in many species.
Responses to both low fluences (LF) and very low fluences
(VLF)
(Mandoli and Briggs, 1981; Kronenberg and Kendrick,
1986) have been reported for light induced germination
(references in VanDerWoude, 1989). For seed batches that
respond in the LF range only, relatively long exposures
(typically on the order of minutes) to light treatments that
form between 2 and 86 % of Pfr are required to trigger
germination, and the potential response is fully reversible
by far-red (FR). In Datura ferox L., this type of
photoresponse is typical of batches of freshly collected
seeds or seeds that have been maintained in dry storage, and
is known to be mediated by a phytochrome pool that is highly
stable in the Pfr form (Casal et al., 1991).
Light sensitivity in seeds of some species can be
increased dramatically, leading to VLF responses, by
treatments such as pre-incubation at low (VanDerWoude and
Toole, 1980; Cone et al., 1985; VanDerWoude, 1985) or high
temperature (Cone et al., 1985; Taylorson and Dinola, 1989),
treatment with ethanol (VanDerWoude, 1985), and GA, (Rethy
et al., 1987). VLF responses are saturated by light pulses
that would establish <10-2% of Pfr, and are therefore not
94
reversible by FR. In D. ferox, the induction of VLF
responses takes place naturally while the seeds are buried
in the soil, apparently without any requirement for chilling
temperatures (Scopel et al., 1991a). The acquisition of
sensitivity to VLFs appears to play a critical role in the
mechanisms whereby buried weed seeds detect the occurrence
of soil disturbances in arable land (Scopel et al., 1991a,
1993; Ballare et al., 1992), but the mechanism of
sensitization is unknown.
An unsolved problem is how the LF- and VLF-response
mechanisms differ at the molecular level. VanDerWoude (1985,
1987, 1989) has proposed a model of phytochrome action that
may account for the existence of VLF and LF responses on the
basis of the dimeric nature of phytochrome's quaternary
structure (Jones and Quail, 1986; Brockmann et al., 1987).
The central idea in VanDerWoude's model is that LF responses
are triggered by a complex formed by an unknown (membrane
associated) receptor "X" and a phytochrome dimer that has
both chromophores in the Pfr form (i.e. X-Pfr:Pfr), whereas
VLF responses are triggered by the association between X and
the heterologous phytochrome dimer (i.e. X-Pfr:Pr).
Conditions that shift seed germination from a LF to a VLF
response would do so by altering the activity (lateral
membrane mobility) of the X-Pfr:Pr effector complex
(VanDerWoude, 1985, 1987), or by altering the relative
95
affinities of X for the two active phytochrome dimers
(Pr:Pfr and Pfr:Pfr)
(De Petter et al., 1988). Models that
do not require phytochrome to be a dimer have also been
suggested to explain the existence of LF- and VLF-responses
(Cone et al., 1985), and the idea of a single "primary
reactant" (i.e. a single "X") for phytochrome appears to be
inconsistent with data obtained in studies of the action of
phytochrome on the appearance of glutamate synthase (Hecht
and Mohr, 1990).
In some systems, sensitization toward VLFs can be
obtained by treatments with hormones, such as IAA in R-
regulated coleoptyle elongation (Shinkle and Briggs, 1984),
and GA, in light promoted germination of Kalanchoe seeds
(Rethy et al., 1987). However, it is unclear whether the
variations in endogenous hormone levels triggered by
developmental or environmental signals actually modulate the
light sensitivity of these systems under natural conditions.
In D. ferox seeds, germination responses toward VLFs can be
induced by incubation in a WSA at constant 25°C (Scopel et
al. 1991b), and previous work has indicated that WSA
treatments cause a reduction in the endogenous levels of
inhibitors, including ABA (De Miguel, 1980). Although this
association is suggestive, neither the effects of ABA on VLF
responses nor the natural dynamics of endogenous ABA in
buried seeds have been previously investigated.
96
In the experiments reported here we have studied the
natural variations in ABA levels during seed burial and
investigated whether these variations may play a causal role
in shifting seed populations between the LF- and VLF-
responding states. We used the arable weed D. ferox as a
test material because detailed information on changes in
fluence response curves for phytochrome controlled
germination during burial is already available, and because
the VLF mechanism appears to play an important role in the
germination ecology of this species (Scopel et al., 1991a).
97
MATERIALS AND METHODS
Seed sources
Seeds of the summer annual weed Datura ferox L.
("chamico"; "chinese thornapple") were harvested in the fall
of 1989 and 1990 from plants growing in soybean fields in
Lobos, Buenos Aires, Argentina. Only seeds with dark seed
coats (Ballare et al., 1988) were used in these experiments.
The seeds were air dried and stored in dark jars at 20 °C
until needed. For the germination tests, the seeds were
placed in 10 x 10 x 1-cm plastic boxes on one layer of
blotting paper saturated with 6.5 mL of distilled water or
ABA solution.
Burial experiments
Plastic mesh bags containing 50 seeds each were either
buried at a depth of 7 cm in a seed bed adjacent to the
laboratory in Corvallis, OR, USA, on 10 May 1991; or buried
at a depth of 5 cm in 10 x 10-cm plastic pots filled with
commercial garden soil and placed in a growth chamber. The
seed bed was kept free of weeds and was exposed to actual
weather conditions. Temperature at the depth of seed burial
was > 13°C and < 33°C during the spring and summer months.
Soil temperature at seed depth in the growth chamber
98
experiment fluctuated between 20°C (15 h) and 30°C (9 h).
The pots were irrigated to maintain the soil water content
near 70 % of field capacity.
Germination tests were performed prior to seed burial
in order to obtain the initial status of the seed
population. At each sampling date (see Figs. 4.2 and 4.3),
seeds were exhumed in absolute darkness. A light proof
device was used to retrieve seed bags buried outdoors. A
very dim green light (Scopel et al., 1991a) was used to
handle the seed samples in the dark room. The seeds were
rapidly rubbed with cotton cloth to remove adhered soil
particles, and either stored at
80°C for ABA determination
or immediately transferred to the germination boxes
containing blotting paper moistened with distilled water and
exposed to the light treatments (see below). After
irradiations, seeds were incubated for 120 h at 20°C/30°C.
For each sampling date, three replicate samples of 50 seeds
each were used to test germination. Except for the lack of
exposure to R or FR light, the dark controls were subjected
to identical manipulation.
Water saturated atmosphere
Incubation in water saturated atmospheres (WSAs) was
carried out as described by De Miguel (1980) at 25°C in
99
darkness. Seeds were retrieved from the WSA chamber in
absolute darkness, rapidly rubbed between cotton cloths and
processed and incubated as indicated for the burial
experiments.
Light sources and assessment of light sensitivity
R was provided by a bank of four fluorescent tubes
(Sylvania, Danvers, MA). Irradiation time was 17 min; total
fluence = 16,198 Amol m-2;
max =
660 nm; calculated % Pfr in
the seeds after filtering by the seed coats (Scopel et al.,
1991a) = 39. FR was obtained by filtering the light from a
250-W incandescent bulb through 5 cm of water and a RG9
filter (Schott, PA, USA); irradiation time = 12 sec; total
fluence = 10.4 Amol m-2; calculated % Pfr = 9.5 10-2. The
germination response to saturating R was considered the
maximum attainable light-induced germination response. The
germination response to FR was used to estimate the
magnitude of the VLF response, as the amount of Pfr formed
by the FR treatment would be enough to saturate a VLF
response but approximately two orders of magnitude lower
than the required to trigger a LF response.
ABA quantitation and manipulation
For each determination of endogenous ABA levels, at
100
least two samples (between 0.7 and 1.5 g fresh weight per
sample, approximately 35 to 55 seeds) of intact seeds of D.
ferox were used. The seeds were powdered in a liquid
nitrogen-chilled mortar together with 2H-ABA (gift from Dr.
JG Buta, USDA, Beltsville, MD, USA)
(1 ug g-1 sample) as an
internal standard and 50,000 dpm DL-cis,trans-[G-21-1]ABA
(66.4 Ci mmo1-1, Amersham) as radiotracer. The powder was
washed with 10 mL of 70% acetone (aqueous, v/v) and allowed
to equilibrate overnight at 4°C. The extract was then
filtered, rinsed 3 times with 5 mL of 70% acetone and dried
in a rotary evaporator. The residue was reconstituted with 5
mL of acidified water (pH 3.0) and partitioned 3 times with
5 mL aliquots of ethyl acetate in a separatory funnel. The
organic phase was saved in each extraction, pooled, dried in
vacuo, resuspended in 250 AL of 30:1:69 methanol:acetic
acid:water (v/v/v) and injected into a Beckman HPLC equipped
with a 110B pump and 163 variable wavelength detector for
further purification. Absorbance was monitored at 264 nm.
Reversed-phase chromatography was performed using a Supelco
Supelcosil LC-18 3 Am
(3.3 cm x 4.6mm ID) column preceded
by a Supelguard LC-18 5 Am (2cm x 4.6mm) guard. The samples
were run isocratically with 30:1:69 methanol:acetic
acid:water for 20 min at 1 mL min-1. The radioactive
fractions collected from the HPLC were pooled, reduced to
dryness, resuspended in 100 AL of methanol and methylated
using ethereal diazomethane (Cohen, 1984). The methylated
101
sample was dried under nitrogen, dissolved in 35 AL of ethyl
acetate and kept at
80 °C until analyzed by GC-SIM-MS. GC­
MS was performed using a Hewlett-Packard 5890 GC and a
Hewlett-Packard 5971A mass selective detector coupled to the
GC using SIM with a dwell time of 25 ms for each ion. The
selected ions monitored were m/z 190 and 193. The levels of
endogenous ABA were calculated from data similar to those
shown in Fig. 4.1 using an isotope dilution equation derived
by Cohen et al. (1986). The calculated R value used in the
equation was 2.844.
In the ABA addition experiments, seeds retrieved from
the soil or from the WSA were placed in direct contact with
the ABA (Sigma) solution in the germination box and
immediately exposed to the light treatments. Four ABA levels
were tested: ABA 0 mM, 0.02 mM, 0.03 mM and 0.05 mM, and
between 3 and 17 replicate seed boxes of 50 seeds each were
used in the germination tests. A time course of seed
imbibition after transfer to the germination box indicated
that seed FW plateaued at about 10 h after sowing. Radicle
protrusion is not observed until 48 h after irradiation,
which suggested that under this experimental protocol ABA
was allowed ample time to interact with the transduction of
the phytochrome signal.
102
RESULTS
Light sensitivity increases and ABA decreases during burial
Seeds of D. ferox were buried outdoors in Spring 1991.
At the time of burial, seeds required R light to germinate
(Fig. 4.2). The germination response to short pulses of FR
(calculated % Pfr = 9.5 10-2), which was initially
negligible, increased dramatically during the first three
months of burial, and remained high until the end of the
experiment. This observation is interpreted as a natural
induction of the VLF mechanism, and is consistent with
previous results (Scopel et al., 1991a). Under these
experimental conditions we also noticed changes in the
levels of dark germination in the exhumed seeds during
burial. These changes were not always coincident with the
changes in FR (VLF) responsiveness, and their expression was
influenced by the incubation conditions of the retrieved
seed samples (see below and Scopel et al., 1991a). ABA
levels in the seeds dropped drastically during the initial
three months and remained low throughout the remaining
burial period (Fig. 4.2).
Seeds buried in soil containers and maintained in a
growth chamber at alternating 20 °C /30 °C also showed a
transient increase in the responsivity to FR (Fig. 4.3).
103
Compared with the outdoor-burial experiment, the kinetics of
VLF-response induction in the growth chamber was slower and
the response itself was of reduced magnitude. Dark
germination was not observed in exhumed seeds of this
experiment. There was a large reduction of endogenous ABA
during the initial phase of burial, and the ABA drop clearly
preceded the increase in responsivity toward FR pulses (Fig.
4.3). During June there was a slight increase in ABA levels,
which preceded the drop in FR responsivity observed during
the last part of this experiment. The increase in ABA during
June might be associated with an accidental failure of the
watering system and consequent partial drying of the soil in
late May.
Exogenous ABA preferentially inhibits FR-induced germination
The majority of the seeds retrieved from the soil in
October (five months of burial) were induced to germinate by
a short FR pulse followed by alternating 20°C/30°C (Figs.
4.2 and 4.4A). Addition of ABA to the incubation medium
reduced both R- and FR-(VLF-)induced germination, but the
depression of the latter was of much greater magnitude and
occurred at lower ABA concentrations (Fig. 4.4A).
By the end of November (six months of burial), most of
the seeds retrieved from the soil germinated in darkness
104
when incubated at 20°C/30°C (Fig. 4.2). Interestingly,
incubation at constant 25°C (instead of alternating
20°C/30°C) after the light pulses appeared to restore the
light requirement without affecting R- or FR-induced
germination. As in the October experiments, addition of ABA
caused a drop in FR-induced germination relative to R-
induced germination (Fig. 4.4B).
Incubation in a WSA at 25°C induced FR-responsivity in
D. ferox seeds (Fig. 4.5 and Scopel et al., 1991b), although
to a somewhat reduced extent compared with soil burial (cf.
Fig. 4.4). The effect of FR on germination, relative to the
effect of R was strongly reduced by exogenous ABA (Fig.
4.5).
Relationship between VLF response and endogenous ABA levels
The importance of the VLF response (estimated from the
effect of FR on germination) relative to germination induced
by saturating R pulses was plotted against the concentration
of endogenous ABA (Fig. 4.6) for seed samples that had been
either buried or exposed to WSAs, and that were incubated in
distilled water or ABA solutions before the light exposure.
For each data set there was a trend of reduced VLF response
relative to the maximum light response with increasing
endogenous ABA concentration (Fig. 4.6); however, there was
105
considerable variation among data sets in the form of the
relationship. This variation was specially noticeable when
comparing experiments where the various ABA levels were
generated over time (burial or WSAs) with those in which an
initially low level of ABA was artificially increased by
addition of ABA to the incubation medium (WSAs + ABA). In
this latter case the levels of seed ABA were higher and the
apparent response-curve flatter.
106
DISCUSSION
Our results indicate that (a) endogenous ABA levels
decrease during seed burial preceding or concomitantly with
the induction of the VLF germination response (Figs. 4.2 and
4.3);
(b) seed exposure to WSA, a treatment known to reduce
ABA levels in D. ferox We Miguel, 1980, and this report)
also renders the seeds sensitive to VLF (Fig. 4.5);
(c)
addition of ABA to seed incubation medium has a greater
negative impact on the VLF response than on the response to
saturating R or on the level of dark germination (Figs. 4.4
and 4.5). These observations are consistent with the
hypothesis that the drop in ABA levels during burial plays a
role in inducing or unmasking the VLF mechanism in D. ferox
seeds.
Previous studies have shown that, although ABA plays a
role in inducing seed dormancy and accelerating seed
maturation (Karssen et al., 1983), ABA levels and depth of
dormancy are not always correlated in mature seeds (Black,
1991 and references therein). Addition of ABA to mature,
non-dormant seeds frequently inhibits germination. In
Brassica rapa seeds, ABA inhibition is the result of reduced
embryo growth (water uptake), which in turn is the
consequence of ABA inhibition of cell wall loosening
(Schopfer and Plachy, 1985). In intact, R-requiring seeds of
107
D. ferox, exogenous ABA inhibits germination (De Miguel,
1980). In isolated embryos incubated in a mannitol solution,
addition of ABA to the medium restricts their growth
(Sanchez and De Miguel, 1985). These changes in the capacity
of the embryo to grow against an osmotic block might be
significant for radicle protrusion in seeds that, like those
of D. ferox, have dormancy imposed by the enclosing tissues
(see Sanchez et al., 1986, 1990 for further discussions). On
the other hand, the degradation of endosperm cell walls, and
the concomitant reduction of mechanical resistance to
radicle protrusion, appear to be critical requisites for
germination in D. ferox (Sanchez et al., 1986, 1990). This
degradation most likely involves increased mannanase and
mannosidase activities (Sanchez et al., 1987, 1990), an
enzyme system that is known to be inhibited by ABA in
(germinated) lettuce seeds (Dulson et al., 1988).
In our experiments with intact D. ferox
seeds,
addition of ABA to the incubation medium inhibited dark and
R-induced germination. However, it is clear that endogenous
ABA and the levels of R-induced or dark germination are not
correlated within the range of ABA concentrations measured
in the seed burial experiments (Figs. 4.2 and 4.3). Also,
the addition of ABA preferentially inhibited FR-induced
germination (compared with R-induced germination)
(Figs. 4.4
and 4.5). A larger inhibitory effect of ABA on FR- as
108
compared with R-induced germination was also reported by
DeGreef et al.
(1989), who worked with Kalanchoe seeds
sensitized to VLFs by GA,. These results give support to the
idea that the mechanism whereby ABA reduces the responsivity
to VLF is, at least at some point, independent of the effect
of ABA on light-saturated and dark germination. On the other
hand, the lack of a single relationship between endogenous
ABA and VLF responsivity (Fig. 4.6) suggests that a simple
model where the bulk, current level of endogenous ABA is the
sole determinant of the expression of the VLF mechanism is
untenable.
Comparisons between the kinetics of ABA loss and VLF-
response induction in buried seeds (Figs. 4.2 and 4.3)
suggest that, if there is a causal link between the two, the
increase in light sensitivity is not an immediate response
to the ABA drop. Preparatory processes that require the ABA
level to be below a critical threshold might take place
during the period that precedes the increase in responsivity
toward FR. Several genes are known to be negatively
regulated by ABA (Heterington and Quatrano, 1991), and the
lag period might represent the time required by the seeds to
produce a specific receptor for the postulated Pfr:Pr dimer
or a reaction partner for the hypothetical Pfr:Pr-X effector
complex. On the other hand, the ABA addition experiments
(Figs. 4.4 and 4.5), indicate that ABA may have a more
109
immediate effect preventing VLF responses. That ABA has
effects at multiple levels in plant cells is well
established (Hetherington and Quatrano, 1991), and rapid
influences, e.g. on membrane functions, are well documented
although the primary site of ABA action (i.e. lipids,
proteins) is unknown (Hetherington and Quatrano, 1991).
Membrane properties and the mobility of the Pfr:Pr-X
effector play a central role in VanDerWoude's model, and
these might be targets for ABA action. Obviously, our
ability to identify processes that are likely to be affected
by ABA is severely limited by our lack of understanding of
the VLF and LF mechanisms at the molecular level.
Acknowledgments
We are most grateful to Dr. Rodolfo A. Sanchez for
useful discussions and for providing the seeds of Datura
ferox and to Dr. Jerry Cohen for making the Beltsville
facilities available to us and for his expert advise on ABA
determination. We also wish to thank Ms. Yuen Yi Tam and Mr.
Jarco Moreno for practical assistance, Mrs. Gretchen Bracher
for preparing the figures, and Dr. Frank Sorensen and
members of the Pacific Northwest USDA-FS Research Station
for providing the field site and growth chamber facilities
in Corvallis.
110
REFERENCES
Ballare CL, Scopel AL, Ghersa CM, Sanchez RA (1988) The fate
of Datura ferox seeds in the soil as affected by
cultivation, depth of burial and degree of maturity.
Annals of Applied Biology 112: 337-345
BallarA CL, Scopel AL, Sanchez RA, Radosevich SR (1992)
Photomorphogenic processes in the agricultural
environment. Photochemistry and Photobiology 56: 777­
788
Black M (1991) Involvement of ABA in the physiology of
developing and mature seeds. In WJ Davies, HG Jones,
eds, Abscisic Acid. Physiology and Biochemistry. Bios
Sientific Publishers, Oxford, pp 99-124
Brockmann J, Rieble S, Kazarinova-Fukshansky N, Seyfried M,
Schafer E (1987) Phytochrome behaves as a dimer in
vivo. Plant, Cell and Environment 10: 105-111
Casal JJ, SAnchez RA, Di Benedetto AH, De Miguel LC (1991)
Light promotion of seed germination in Datura ferox is
mediated by a highly stable pool of phytochrome.
Photochemistry and Photobiology 53: 249-254
Cohen JD (1984) Convenient apparatus for generation of small
amounts of diazomethane. Journal of Chromatography 303:
193-196.
Cohen JD, Baldi BG, Slovin JP (1986) 13C,-[Benzene ring]
indole-3-acetic acid. Plant Physiology 80:14-19
111
Cone JW, Jaspers PAPM, Kendrick RE (1985) Biphasic fluence­
response curves for light induced germination of
Arabidopsis thaliana seeds. Plant, Cell anf Environment
8: 605-612
De Greef JA, Fredericq H, Rethy R, Dedonder A, De Petter E,
Van Wiemeersch L (1989) Factors affecting the
germination of photoblastic Kalanchoe seeds. In RB
Taylorson, ed, Recent Advances in the Development and
Germination of Seeds. Plenum Press, New York, NY, pp
241-260
Be Miguel LC (1980) Changes in the levels of endogenous
inhibitors during dormancy breakage in Datura ferox L.
seeds. Z Pflanzenphysiol 96: 415-421
De Petter E, Van Wiemeersch L, Rethy R, Dedonder A,
Fredericq H, De Greef J (1988) Fluence-response curves
and action spectra for the very low fluence response
for the induction of Kalanchoe seed germination. Plant
Physiology 88: 276-283
Dulson J, Bewley JD, Johnston RN (1988) Abscisic acid is an
endogenous inhibitor in the regulation of mannanase
production by isolated lettuce (Lactuca sativa cv Grand
Rapids) endosperms. Plant Physiology 87: 660-665
Hecht II, Mohr H (1990) Relationship between phytochrome
photoconversion and response. Photochemistry and
Photobiology 51: 369-373
Hetherington AM, Quatrano RS (1991) Mechanisms of action of
112
abscisic acid at the cellular level. New Phytologist
119: 9-32
Jones AM, Quail PH (1986) Quaternary structure of 124­
kilodalton phytochrome from Avena sativa L.
Biochemistry 25: 2987-2995
Karssen CM, Brinkhorst-van der Swan DLC, Breekland AE,
Koornneef M (1983) Induction of dormancy during seed
development by endogenous abscisic acid: studies on
abscisic acid deficient genotypes of Arabidopsis
thaliana (L.) Heynh. Planta 157: 158-165
Kronenberg GHM, Kendrick RE (1986) The physiology of action.
In RE Kendrick, GHM Kronenberg, eds, Photomorphogenesis
in Plants. Martinus Nijhoff, Dordrecht, pp 99-114
Mandoli DF, Briggs WR (1981) Phytochrome control of two low­
irradiance responses in etiolated oat seedlings. Plant
Physiology 67: 733-739
Rethy R, Dedonder A, De Petter E, Van Wiemeersch L,
Fredericq H, De Greef J, Steyaert H, Stevens H (1987)
Biphasic fluence-response curves for phytochrome­
mediated Kalanchoe seed germination. Plant Physiology
83: 126-130
Sanchez RA, De Miguel LC (1985) The effect of red light, ABA
and K+ on the growth rate of Datura ferox embryos and
its relations with the photocontrol of germination.
Botanical Gazzette 146: 472-476
Sanchez RA, De Miguel LC, Mercuri 0 (1986) Phytochrome
113
control of cellulase activity in Datura ferox seeds,
its relationship with germination. Journal of
Experimental Botany 37: 1574-1580
Sanchez RA, Sunell L, Labavitch JM, Bonner BA (1987) Changes
in the micropylar region of the endosperm, before
radicle protrusion in the seeds of two Datura species
(abstract No. 711). Plant Physiology 83: S118
Sanchez RA, Sunell L, Labavitch JM, Bonner BA (1990) Changes
in the endosperm cell walls of two Datura species
before radicle protrusion. Plant Physiology 93: 89-97
Schopfer P, Plachy C (1985) Control of seed germination by
abscisic acid. III. Effect on embryo growth potential
(minimum turgor pressure) and growth coefficient (cell
wall extensibility) in Brassica napus L. Plant
Physiology 77: 676-686
Scopel AL, Ballard CL, Radosevich SR (1993) Photostimulation
of seed germination during soil tillage. New
Phytologist, in press.
Scopel AL, Ballard CL, Sanchez RA (1991a) Induction of
extreme light sensitivity in buried weed seeds and its
role in the perception of soil cultivations. Plant,
Cell and Environment 14: 501-508
Scopel AL, Ballard CL, Sanchez RA (1991b) Very-Low-Fluence­
responses in light promoted germination of buried weed
seeds. Implications for the perception of soil
cultivation. Beltsville Symposium XVI.
114
Photomorphogenesis in Plants: Emerging Strategies for
Crop Improvement. Book of Abstracts, pp 13. College
Park, MD
Taylorson RB, Dinola L (1989) Increased phytochrome
responsiveness and a high-temperature transition in
Barnyardgrass (Echinochloa crus-galli) seed dormancy.
Weed Science 37: 335-338
VanDerWoude WJ (1985) A dimeric mechanism for the action of
phytochrome: evidence from phototermal interactions in
lettuce seed germination. Photochemistry and
Photobiology 42: 655-661
VanDerWoude WJ (1987) Application of the dimeric model of
phytochrome action to high irradiance responses. In M
Furuya, ed, Phytochrome and Photoregulation in Plants.
Academic Press, London, pp 249-258
VanDerWoude WJ (1989) Phytochrome and sensitization in
germination control. In RB Taylorson, ed, Recent
Advances in the Development and Germination of Seeds.
Plenum Press, New York, NY, pp 181-189
VanDerWoude WJ, Toole VK (1980) Studies on the mechanism of
enhancement of phytochrome-dependent lettuce seed
germination by prechilling. Plant Physiology 66: 220­
224
115
40
TIC of data: AS4LAB2.D
30
20
10
0
40
Ion 190
Area 407925
30
20
10­
0
20
Ion 193
Area 43152
10­
,...A...................A/\".
6.2
6.4
6.6
6:8
7.0
7.2
7.4
Time (min.)
Figure 4.1 Total (upper panel) and selected ion
chromatograms of a methylated HPLC purified sample of ABA
from intact seeds of D. ferox. The ions at m/z 190 and 193
represent the base peaks of MeABA and the deuteriated
internal standard, respectively.
116
100 -
0
- 150
vi
75-
rn
a
0
CO
ca
E 50
- 75
_
cg
rn
25­
0
0
May
Aug
Sep
Oct
Nov
Dec
Date
Figure 4.2 Time courses of dark germination, light
sensitivity and ABA levels in D. ferox seeds buried
outdoors. Seeds were buried in May, exhumed in darkness at
the indicated dates, rapidly rubbed with cotton cloth to
remove adhered soil particles, and either stored at -80°C
for ABA determination or immediately transferred to blotting
paper moistened with distilled water and exposed to the
light treatments. After irradiation, seeds were incubated
for 120 at 20°C/30°C. The calculated Pfr level established
by the light treatments (see "Materials and Methods") was 39
t after R and 10-2 t after FR. Each datum point is the mean
of three replicate samples of 50 seeds each (germination) or
two samples of 0.7-1.5 g FW of intact seeds each (ABA) ± 1
SE (when larger than the symbol).
117
200
R
O FR
Dark
100 -
150
t
t
.....---"*...ifroe..0".
100
t
-6-
'a-
ei)
75­
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'.
.
0-0--0
I
Mar
1
0
111
I
I
Apr
I
May
I
1
Jun
Jul
Aug
Date
Figure 4.3 Time courses of dark germination, light
sensitivity and ABA levels in D. ferox seeds buried in a
growth chamber at 20°C/30°C. Seeds were buried in March,
exhumed in darkness at the indicated dates, and processed as
indicated in the legend to Fig. 4.2. Each datum point is the
mean of three replicate samples of 50 seeds each
(germination) or two samples of 0.7-1.5 g FW of intact seeds
each (ABA) ± 1 SE (when larger than the symbol).
118
1
0
0.01
0.02
0.03
0.04
0.05
Exogenous ABA (mM)
Figure 4.4 Effect of exogenous ABA on light sensitivity of
D. ferox seeds exhumed after five (A) or six (B) months of
burial outdoors. Seeds were retrieved in absolute darkness,
rapidly rubbed to eliminate adhered soil particles, and
transferred to plastic boxes containing blotting paper
saturated with ABA solutions of the indicated
concentrations. Seeds were then immediately exposed to the
light treatments and incubated for 120 h at 20°C/30°C (A) or
at 25 °C (B). The calculated Pfr level established by the
light treatments (see "Materials and Methods") was 39 %
after R and 10-2 % after FR. Each datum point is the mean of
three to eight replicate samples of 50 seeds each ± 1 SE.
Statistical analysis in Appendix 2.
119
0
0.01
0.02
0.03
0.04
0.05
Exogenous ABA (mM)
Figure 4.5 Effect of exogenous ABA on light sensitivity of
D. ferox seeds that had been kept for three weeks in a WSA.
Seeds were retrieved from the WSA chamber in absolute
darkness, rapidly rubbed between cotton clothes, processed
as indicated in the legend to Fig. 4.4, and incubated for
120 h at 20°C/30°C. The calculated Pfr level established by
the light treatments (see "Materials and Methods") was 39 %
after R and 9.5 10-2 % after FR. Each datum point is the
mean of 10 to 17 replicate samples of 50 seeds each ± 1 SE.
Statistical analysis in Appendix 2.
120
O
O
T- 100-.
Buried outdoors
Buried at 20-30°C
0 WSA
IF
O WSA + ABA
co
1?
cc
75­
.-.--.-,
_Ne
cis
cc113
u_
50­
c0
fs
c_
coh­
25­
(5
0
I
I
100
200
1
300
400
Endogenous ABA (gg g-1 FW)
Figure 4.6 Relationships between VLF responsivity and
endogenous ABA levels in whole seeds of D. ferox. The
different data sets correspond to seed samples that had
spent variable periods of time in the soil or were exposed
to WSAs, and that were incubated in distilled water or ABA
solutions prior to the light exposure. The primary data used
to construct this figure were presented in Figs. 4.2 (Buried
outdoors), 4.3 (Buried at 20°C/30°C); germination data for
the WSA data sets were presented in Fig. 4.5 (WSA + ABA) or
were obtained from germination tests carried out before and
after the exposure to WSA (WSA). The ratio (FR-dark)/(R­
dark) was calculated with the average germination induced by
each irradiation treatment. Endogenous ABA levels were
obtained as indicated in Materials and Methods.
121
CHAPTER 5
SUMMARY AND OUTLOOK
The main theme of this thesis is the photocontrol of
seed germination in arable land. I have approached this
problem at different scales, from the field experiments
using large plots and farm equipment, to the dark-room
experiments aimed at understanding the physiological basis
of changes in light sensitivity. This multi-scale approach
was useful to narrow the gaps in the understanding of how
the photophysiology of seeds changes in the natural
environment and how this change affects the responses of
seed banks to man-made perturbations.
Most previous studies on the photocontrol of seed
germination have been accomplished with seeds that display
R/FR reversible photoresponses (i.e. LF responses). The
results presented in Chapter 2 demonstrated, for the first
time, that seeds of an arable weed (Datura ferox) naturally
acquire the ability to respond to VLFs after burial. VLF-
responding seeds are roughly 10,000 times more sensitive to
122
light than LF-responding seeds, suggesting that care should
be taken when attempting to predict the photoresponses of
buried seeds from laboratory studies.
Responses to VLFs had been reported previously for
several plants systems, including seeds exposed to a variety
of artificial pretreatments under laboratory conditions.
However, the ecological significance of the very high light
sensitivity is not immediately obvious, and VLF responses
have largely been regarded as laboratory artifacts. The
experiments described in Chapter 2 and 3 demonstrated that
not only is the ability to respond to VLFs induced naturally
during burial, but also that the high light sensitivity is
expressed in seeds buried in the soil under field
conditions.
These results call attention to two important points.
First, what is the ecological significance (i.e.
"evolutionary purpose") of the shift in light sensitivity
and what are the managerial implications for agriculture.
Second, what is the physiological basis of the induction of
the VLF mechanism. Both questions were addressed in this
thesis. What follows is a brief summary of the major
findings and their likely implications.
One of the challenges faced by ecologists is to scale
123
up the wealth of physiological information to understand and
predict the responses of complex ecological systems.
Physiological studies on seed germination typically focus on
the effect of a single factor under well defined
experimental conditions, which are set deliberately to
exclude interactions and other sources of "noise". Yet, it
is the product of such interactions that seed ecologists
attempt to predict and agronomists seek to manipulate. In
Chapter 2,
I proposed that a critical role of the VLF
response is to allow seeds to detect split-second exposures
to sunlight when the surrounding soil is disturbed by
tillage operations, and that these short light pulses are
critical germination cues for species that invade arable
lands. This hypothesis was consistent with the results of
field experiments in which seed samples were exposed to
relatively simple perturbations. However, many previous
studies have demonstrated that light interacts with other
environmental factors (e.g. temperature) in the control of
seed germination. Several of these factors are altered when
the soil is cultivated, making the quantitative importance
of light signals difficult to predict. Moreover, seeds of
several weedy species or ecotypes can readily germinate in
darkness, at least under some conditions, adding a further
complication to the prediction of seed bank responses.
The experiments presented in Chapter 3 were designed to
124
elucidate the role of light perception by seeds during soil
disturbances and to assess the significance of short light
pulses on germination induction. Soil cultivation with farm
equipment during daytime enhanced weed seed germination
between 70 and 400 % above those levels recorded for the
same set of cultivations performed during the night.
Covering the implements during daytime cultivation, which
prevented sunlight from reaching the soil during the actual
disturbance, reduced the germination of dicotyledonous
species to levels that were similar to those obtained after
nighttime cultivation. On the other hand, nighttime
cultivation under strong artificial light mounted on the
harrow equipment significantly promoted seedling emergence
compared with the dark control. These findings suggest that
pulse-like irradiations perceived by the seeds during soil
disturbance play an important role in triggering seed
germination in disturbed soil. Calculations indicate that a
mechanism sensitive to VLFs is required for the
photodetection of these short exposures to sunlight,
pointing to a critical ecological role for the VLF
phytochrome response in seeds. More research is required to
test the general validity of these conclusions in other
cropping systems and under different ecological scenarios,
as discussed in Chapter 3. Given the potential practical
applications of manipulating the light environment to
influence seed germination, agricultural and ecological
125
research in this direction seems well warranted.
The experiments presented in Chapter 4 were designed to
test the potential role of changes in endogenous levels of
abscisic acid (ABA) in the shifts of light sensitivity
experienced by seeds when they are buried in the soil. In
general, the results showed that (i) ABA levels decrease
naturally during burial, concomitantly or preceding the
induction of VLF responsiveness in seeds,
(ii) the relative
importance of the VLF, compared with the LF, and the level
of endogenous ABA are inversely related, and (iii) the
magnitude of the VLF relative to the LF can be reduced by
experimental ABA addition to the seed incubation media
These results suggest that the drop in ABA is causally
involved in inducing or unmasking the VLF mechanism in
seeds. Although interactions between light and growth
regulators have been demonstrated in several systems this
would be the first case in which the natural changes in the
levels of a growth regulator have been related to the
appearance of the VLF mechanism. If ABA does indeed play a
role modulating light sensitivity the obvious next question
is by which mechanism(s). At this point our ability to
identify targets for ABA action is severely limited by our
lack of understanding of how LF and VLF responses differ at
the molecular level.
126
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APPENDICES
140
APPENDIX 1
Statistical analyses of data presented in Chapter 3
Figure 3.1 Statistical analysis (using SAS algorithms)
Inc. 1987) of data presented in Fig. 3.1.
(SAS Institute
General Linear Models Procedure
Class Level Information
Class
Levels
BLOCK
10
TREAT
3
Values
1 2 3 4 5 6 7 8 9 10
LA LS N
Number of observations in data set = 30
NOTE: LA = Daytime cultivation (Amaranthus retroflexus); LS = Daytime
cultivation (Solanum spp.); N = Nighttime cultivation (control =l)
General Linear Models Procedure
Dependent Variable: RATIO
Source
DF
BLOCK
TREAT
2
Source
DF
BLOCK
TREAT
Error
Corr.Tot
9
9
2
18
29
Type I SS
86.7836800
117.4166067
Mean Square
F Value
9.6426311
58.7083033
1.07
6.54
Type III SS
Mean Square
F Value
86.7836800
117.4166067
161.5286600
365.7289467
9.6426311
58.7083033
8.9738144
1.07
6.54
R-Square
0.558338
C.V.
79.27750
Root MSE
2.995633
Pr > F
0.4257
0.0073
Pr > F
0.4257
0.0073
RATIO Mean
3.77866667
141
General Linear Models Procedure
Least Squares Means
TREAT
LA
LS
N
RATIO
LSMEAN
Std Err
LSMEAN
HO:LSMEAN=0
5.45100000
4.88500000
1.00000000
0.94730219
0.94730219
0.94730219
0.0001
0.0001
0.3051
Pr >
Pr >
IT1
LSMEAN
Number
1
2
3
IT1 HO: LSMEAN(i)=LSMEAN(j)
i/j
1
2
3
2
1
.
0.6777
0.0038
0.6777
.
3
0.0038
0.0095
0.0095
Fisher's Protected LSD (FPLSD)
FPLSD = tdf,0.05 x [2MSE/r]°.5
FPLSD = 2.11 x (2(8.97)/10)0.5 = 2.826
NOTE: LA (Daytime cultivation, Amaranthus retroflexus) significantly
different from N (nighttime cutivation); LS (Daytime cultivation,
Solanum spp.) significantly different from N (nighttime cutivation).
142
Figure 3.2 Statistical analysis (using SAS algorithms)
Inc. 1987) of data presented in Fig. 3.2.
(SAS Institute
Winter-annual dicots
General Linear Models Procedure
Class Level Information
Values
Class
Levels
BLOCK
6
1 2 3 4 5 6
TREAT
3
L N NL
Number of observations in data set = 18
NOTE: L = Daytime cultivation; N = Nighttime cultivation (control =l); NL
= Nighttime cultivation + Low light
General Linear Models Procedure
Dependent Variable: RATIO
Source
Pr > F
Model
0.0159
Error
Corrected Total
Mean
Square
DF
Sum of
Squares
7
5.54801667
0.79257381
10
17
1.74523333
7.29325000
0.17452333
R-Square
C.V.
Root MSE
0.760706
29.45428
0.417760
F Value
4.54
RATIO Mean
1.41833333
General Linear Models Procedure
Dependent Variable: RATIO
Type I SS
Mean Square
F Value
Source
DF
BLOCK
TREAT
5
2
1.35031667
4.19770000
0.27006333
2.09885000
Source
DF
Type III SS
Mean Square
F Value
BLOCK
TREAT
5
2
1.35031667
4.19770000
0.27006333
2.09885000
1.55
12.03
1.55
12.03
Pr > F
0.2601
0.0022
Pr > F
0.2601
0.0022
143
General Linear Models Procedure
Least Squares Means
TREAT
L
N
NL
RATIO
LSMEAN
Std Err
LSMEAN
HO:LSMEAN=0
2.09500000
1.00000000
1.16000000
0.17054976
0.17054976
0.17054976
0.0001
0.0002
0.0001
Pr >
Pr >
IT1
LSMEAN
Number
1
2
3
IT; HO: LSMEAN(i)=LSMEAN(j)
i/j
1
2
3
2
3
0.0011
0.0031
0.5221
1
.
0.0011
0.0031
.
0.5221
NOTE: To ensure overall protection level, only probabilities associated
with pre-planned comparisons should be used.
Fisher's Protected LSD (FPLSD)
FPLSD = tdC0.05 x [2MSE/r]°.5
FPLSD = 2.262 x [2(0.1745)/6]°*5 = 0.545
NOTE: L (Daytime cultivation) significantly different from N (Nighttime
cultivation); NL (Nighttime cultivation + Low light) not different from
N.
144
Grasses
General Linear Models Procedure
Class Level Information
Values
Class
Levels
BLOCK
6
1 2 3 4 5 6
TREAT
3
L N NL
Number of observations in data set = 18
NOTE: L = Daytime cultivation; N = Nighttime cultivation (control =l);
NL = Nightime cultivation + Low light
General Linear Models Procedure
Dependent Variable: RATIO
Sum of
Squares
DF
Source
5.91768889
0.93102222
6.84871111
7
Model
Error
10
Corr.Tot. 17
R-Square
0.864059
Mean
Square
F Value
0.84538413
0.09310222
C.V.
22.90358
9.08
Root MSE
0.305127
Pr > F
0.0012
RATIO Mean
1.33222222
General Linear Models Procedure
Dependent Variable: RATIO
Source
DF
BLOCK
TREAT
5
2
Source
DF
BLOCK
TREAT
5
2
Mean Square
F Value
0.13631111
5.78137778
0.02726222
2.89068889
0.29
31.05
Type III SS
Mean Square
F Value
Type I SS
0.13631111
5.78137778
0.02726222
2.89068889
0.29
31.05
Pr > F
0.9062
0.0001
Pr > F
0.9062
0.0001
145
General Linear Models Procedure
Least Squares Means
TREAT
L
N
NL
RATIO
LSMEAN
Std Err
LSMEAN
HO:LSMEAN=0
2.13000000
1.00000000
0.86666667
0.12456740
0.12456740
0.12456740
0.0001
0.0001
0.0001
Pr >
ITI
LSMEAN
Number
1
2
3
Pr > ITI HO: LSMEAN(i)=LSMEAN(j)
i/j
1
2
3
2
3
0.0001
0.0001
0.4666
1
.
0.0001
0.0001
.
0.4666
NOTE: To ensure overall protection level, only probabilities associated
with pre-planned comparisons should be used.
Fisher's Protected LSD (FPLSD)
FPLSD = tdC0.05 x [2MSE/r]°'5
FPLSD = 2.262 x [2(0.0931)/6] " = 0.398
NOTE: L (Daytime cultivation) significantly different from N (Nighttime
cultivation); NL (Nighttime cultivation + Low light) not different from
N.
146
Figure 3.3 Statistical analysis (using SAS algorithms)
Inc. 1987) of data presented in Fig. 3.3.
(SAS Institute
General Linear Models Procedure
Class Level Information
Class
Levels
BLOCK
12
TREAT
3
Values
1 2 3 4 5 6 7 8 9 10 11 12
DC GC L
Number of observations in data set = 36
NOTE: DC = Daytime cultivation with the implements covered (Winter­
annual dicots); GC = Daytime cultivation with the implements covered
(Grasses); L = Daytime cultivation (control =l)
General Linear Models Procedure
Dependent Variable: RATIO
Sum of
DF
Source
Squares
Model
Error
Corr.Tot.
1.19266111
0.85396111
2.04662222
13
22
35
R-Square
0.582746
Mean
Square
F Value
Pr > F
2.36
0.0364
0.09174316
0.03881641
C.V.
22.08181
Root MSE
0.197019
RATIO Mean
0.89222222
General Linear Models Procedure
Dependent Variable: RATIO
Source
DF
Type I SS
Mean Square
F Value
Pr > F
BLOCK
TREAT
11
2
0.50755556
0.68510556
0.04614141
0.34255278
1.19
8.82
0.3493
0.0015
Source
DF
Type III SS
Mean Square
F Value
Pr > F
BLOCK
TREAT
11
0.50755556
0.68510556
0.04614141
0.34255278
1.19
8.82
0.3493
0.0015
2
147
General Linear Models Procedure
Least Squares Means
TREAT
DC
GC
L
RATIO
LSMEAN
Std Err
LSMEAN
Pr > ITI
HO:LSMEAN =O
0.69750000
0.97916667
1.00000000
0.05687443
0.05687443
0.05687443
0.0001
0.0001
0.0001
Pr >
LSMEAN
Number
1
2
3
ITI HO: LSMEAN(i)=LSMEAN(j)
1
2
3
2
1
i/j
.
0.0020
0.0011
0.0020
.
3
0.0011
0.7980
0.7980
NOTE: To ensure overall protection level, only probabilities associated
with pre-planned comparisons should be used.
Fisher's Protected LSD (FPLSD)
FPLSD = tdt,0)5 x [2MSE/r]°-5
FPLSD = 2.08 x [2(0.0388)/12] 0'5 = 0.167
NOTE: DC (Daytime cultivation with the implements covered [Winter-annual
dicots]) significantly different from L (Daytime cultivation); GC
(Daytime cultivation with the implements covered [Grasses]) not
significantly different from L (Daytime cultivation)
148
Figure 3.4 Statistical analysis (using SAS algorithms)
Inc. 1987) of data presented in Fig. 3.4.
(SAS Institute
Autumn
General Linear Models Procedure
Class Level Information
Class
Levels
BLOCK
26
TREAT
4
Values
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
22 23 24 25 26
C L NL N
NOTE: C = Daytime cultivation with the implements cover; L = Daytime
cultivation; NL = Nightime cultivation + Strong illumination; N=
Nightime cultivation (control =l)
Number of observations in data set = 104
General Linear Models Procedure
Dependent Variable: RATIO
Sum of
DF
Source
Squares
28
Model
75
Error
Corr.Tot. 103
29.01502692
42.69037212
71.70539904
R-Square
0.404642
Mean
Square
F Value
Pr > F
1.82
0.0213
1.03625096
0.56920496
C.V.
55.26766
Root MSE
0.754457
RATIO Mean
1.36509615
General Linear Models Procedure
Dependent Variable: RATIO
Source
DF
Type I SS
Mean Square
F Value
Pr > F
BLOCK
TREAT
25
3
16.39572404
12.61930288
0.65582896
4.20643429
1.15
7.39
0.3118
0.0002
Source
DF
Type III SS
Mean Square
F Value
Pr > F
BLOCK
TREAT
25
16.39572404
12.61930288
0.65582896
4.20643429
1.15
7.39
0.3118
0.0002
3
149
General Linear Models Procedure
Least Squares Means
TREAT
C
L
NL
N
RATIO
LSMEAN
Std Err
LSMEAN
HO:LSMEAN=0
1.12192308
1.90153846
1.43692308
1.00000000
0.14796114
0.14796114
0.14796114
0.14796114
0.0001
0.0001
0.0001
0.0001
Pr >
I
i/j
1
2
3
4
Ti
Pr >
I
Ti
LSMEAN
Number
1
2
3
4
HO: LSMEAN(i)=LSMEAN(j)
2
3
0.0004
0.1364
0.0294
1
.
0.0004
0.1364
0.5619
.
0.0294
0.0001
.
4
0.5619
0.0001
0.0402
0.0402
NOTE: To ensure overall protection level, only probabilities associated
with pre-planned comparisons should be used.
Fisher's Protected LSD (FPLSD)
FPLSD = tdf.), x [2MSE/r]°.5
FPLSD = 1.960 x [2(0.569)/26]0.5 = 0.410
NOTE: L (Daytime cultivation) is significantly different from N
(Nighttime cultivation); NL (Nighttime cultivation + strong
illumination) is significantly diferent from N; C (Daytime cultivation
with the implements covered) is not significantly different from N.
150
Summer
General Linear Models Procedure
Class Level Information
Class
Levels
BLOCK
24
TREAT
4
Values
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
22 23 24
C L NL N
Number of observations in data set = 96
NOTE: C = Daytime cultivation with the implements cover; L = Daytime
cultivation; NL = Nightime cultivation + Strong illumination; N =
Nightime cultivation (control =l)
General Linear Models Procedure
Dependent Variable: RATIO
Sum of
DF
Squares
Source
Model
Error
Corr.Tot.
26
69
95
71.79830000
46.53616250
118.33446250
R-Square
0.606740
Mean
Square
F Value
Pr > F
4.09
0.0001
2.76147308
0.67443714
C.V.
57.75762
Root MSE
0.821241
RATIO Mean
1.42187500
General Linear Models Procedure
Dependent Variable: RATIO
Source
DF
Type I SS
Mean Square
F Value
Pr > F
BLOCK
TREAT
23
3
57.82426250
13.97403750
2.51409837
4.65801250
3.73
6.91
0.0001
0.0004
Source
DF
Type III SS
Mean Square
F Value
Pr > F
BLOCK
TREAT
23
57.82426250
13.97403750
2.51409837
4.65801250
3.73
6.91
0.0001
0.0004
3
151
General Linear Models Procedure
Least Squares Means
TREAT
C
L
NL
N
RATIO
LSMEAN
Std Err
LSMEAN
Pr > IT1
HO:LSMEAN =O
1.09500000
1.88875000
1.70375000
1.00000000
0.16763516
0.16763516
0.16763516
0.16763516
0.0001
0.0001
0.0001
0.0001
Pr >
ITI
2
3
4
.
0.0013
0.0124
0.6899
1
2
3
4
LSMEAN(i)=LSMEAN(j)
2
3
4
0.0013
0.0124
0.4379
0.6899
0.0004
0.0041
1
i/j
1
HO:
LSMEAN
Number
.
0.4379
0.0004
.
0.0041
Fisher's Protected LSD (FPLSD)
FPLSD = tdf, x [2MSE/r]°.5
FPLSD = 1.960 x [2(0.6744)/24] " = 0.465
NOTE: L (Daytime cultivation) is significantly different from N
(Nighttime cultivation); NL (Nighttime cultivation + strong
illumination) is significantly diferent from N; C (Daytime cultivation
with the implements covered) is not significantly different from N.
152
APPENDIX 2
Statistical analyses of data presented in Chapter 4
Figure 4.4 Statistical analysis (using SAS algorithms)
Inc. 1987) of data presented in Fig. 4.4.
(SAS Institute
A)
General Linear Models Procedure
Class Level Information
Class
Levels
Values
ABA
4
ABO AB2 AB3 AB5
IRRAD
2
FR R
Number of observations in data set = 53
NOTE: ABO = ABA 0 mM; AB2 = 0.02 mM; AB3 = 0.03 mM; AB5 = 0.05 mM; FR =
Far Red; R = Red
General Linear Models Procedure
Dependent Variable: GERM
Source
DF
Model
Error
Corrected Total
45
52
Sum of
Squares
39628.17672
8311.74045
47939.91717
7
R-Square
0.826622
C.V.
17.07994
Mean
Square
5661.16810
184.70534
F Value
Pr > F
30.65
0.0001
Root MSE
13.59063
GERM Mean
79.5707547
General Linear Models Procedure
Dependent Variable: GERM
Source
DF
ABA
IRRAD
3
1
ABA*IRRAD
3
Source
DF
ABA
3
IRRAD
1
ABA*IRRAD
3
Type I SS
26611.83148
5520.67700
7495.66824
Type III SS
24766.46500
8795.18333
7495.66824
Mean Square
F Value
8870.61049
5520.67700
2498.55608
48.03
29.89
13.53
Mean Square
F Value
8255.48833
8795.18333
2498.55608
44.70
47.62
13.53
Pr > F
0.0001
0.0001
0.0001
Pr > F
0.0001
0.0001
0.0001
153
General Linear Models Procedure
Least Squares Means
ABA
GERM
LSMEAN
Std Err
LSMEAN
90.4187500
97.4777778
86.8750000
93.7666667
19.7500000
96.0000000
8.0333333
50.0000000
2.7741766
4.5302115
6.7953172
7.8465564
6.7953172
7.8465564
7.8465564
7.8465564
IRRAD
FR
R
FR
R
FR
R
FR
R
ABO
ABO
AB2
AB2
AB3
AB3
AB5
AB5
Pr >
ITI
HO:LSMEAN=0
LSMEAN
Number
0.0001
0.0001
0.0001
0.0001
0.0057
0.0001
0.3114
0.0001
1
2
3
4
5
6
7
8
General Linear Models Procedure
Least Squares Means
Least Squares Means for effect ABA*IRRAD
Pr > IT1 HO: LSMEAN(i)=LSMEAN(j)
Dependent Variable: GERM
1
2
3
4
5
6
7
8
2
3
4
5
6
7
8
0.1906
0.6316
0.2008
0.6894
0.6840
0.5101
0.0001
0.0001
0.0001
0.0001
0.5059
0.8712
0.3840
0.8414
0.0001
0.0001
0.0001
0.0001
0.0001
0.2650
0.0001
0.0001
0.0001
0.0009
0.0003
0.0055
0.0001
0.0005
1
i/j
.
0.1906
0.6316
0.6894
0.0001
0.5059
0.0001
0.0001
.
0.2008
0.6840
0.0001
0.8712
0.0001
0.0001
.
0.5101
0.0001
0.3840
0.0001
0.0009
.
0.0001
0.8414
0.0001
0.0003
.
0.0001
0.2650
0.0055
.
0.0001
0.0001
.
0.0005
NOTE: To ensure overall protection level, only probabilities associated
with pre-planned comparisons should be used.
NOTE: The interaction between ABA and irradiation (ABA*IRRAD) was
significant at the 0.0001 level. Pairwise comparisons (see "Least Square
Means" Tables) showed no differences between R and FR induced
germination for ABA levels < 0.02 mM, and differences between the two
light treatments for ABA > 0.03 mM.
154
B)
General Linear Models Procedure
Class Level Information
Levels
Class
Values
ABA
4
ABO AB2 AB3 ABS
IRRAD
2
FR R
Number of observations in data set = 23
NOTE: ABO = ABA 0 mM; AB2 = 0.02 mM; AB3 = 0.03 mM; AB5 = 0.05 mM; FR =
Far Red; R = Red
General Linear Models Procedure
Dependent Variable: GERM
Sum of
DF
Squares
Source
Model
Error
Corr.Tot
7
15
22
31495.81738
1620.15787
33115.97525
R-Square
0.951076
Mean
Square
4499.40248
108.01052
C.V.
21.98624
F Value
41.66
Root MSE
10.39281
Pr > F
0.0001
GERM Mean
47.2696174
General Linear Models Procedure
Dependent Variable: GERM
Source
DF
ABA
IRRAD
3
1
ABA*IRRAD
3
Source
DF
ABA
3
IRRAD
1
ABA*IRRAD
3
Type I SS
19806.84961
7636.91068
4052.05709
Type III SS
20271.35492
7237.45267
4052.05709
Mean Square
F Value
6602.28320
7636.91068
1350.68570
61.13
70.71
12.51
Mean Square
F Value
6757.11831
7237.45267
1350.68570
62.56
67.01
12.51
Pr > F
0.0001
0.0001
0.0002
Pr > F
0.0001
0.0001
0.0002
155
General Linear Models Procedure
Least Squares Means
ABA
ABO
ABO
AB2
AB2
AB3
AB3
AB5
AB5
GERM
LSMEAN
Std Err
LSMEAN
HO:LSMEAN=0
89.9666667
93.9333333
11.9000000
85.3000000
7.7336667
53.0333333
0.0001000
20.5333333
6.0002923
6.0002923
6.0002923
6.0002923
6.0002923
6.0002923
7.3488273
6.0002923
0.0001
0.0001
0.0660
0.0001
0.2170
0.0001
1.0000
0.0038
IRRAD
FR
R
FR
R
FR
R
FR
R
Pr >
1T1
LSMEAN
Number
1
2
3
4
5
6
7
8
General Linear Models Procedure
Least Squares Means
Least Squares Means for effect ABA*IRRAD
Pr > 1T1 HO: LSMEAN(i)=LSMEAN(j)
Dependent Variable: GERM
i/j
1
1
2
3
4
5
6
7
8
0.6469
0.0001
0.5905
0.0001
0.0006
0.0001
0.0001
2
3
4
5
6
7
8
0.6469
0.0001
0.0001
0.5905
0.3251
0.0001
0.0001
0.0001
0.6305
0.0001
0.0006
0.0002
0.0002
0.0017
0.0001
0.0001
0.0001
0.2289
0.0001
0.4277
0.0001
0.0001
0.0001
0.3251
0.0001
0.1522
0.0016
0.0470
0.0001
0.3251
0.0001
0.0002
0.0001
0.0001
0.0001
0.6305
0.0002
0.2289
0.3251
0.0001
0.0017
0.0001
0.0001
0.0001
0.4277
0.1522
0.0001
0.0016
0.0470
NOTE: To ensure overall protection level, only probabilities associated
with pre-planned comparisons should be used.
NOTE: The interaction between ABA and irradiation (ABA*IRRAD) was
significant at the 0.0002 level. Pairwise comparisons (see "Least Square
Means" Tables) showed no differences between R and FR induced
germination for ABA levels < 0.02 mM, and differences between the two
light treatments for ABA > 0.02 mM.
156
Figure 4.5 Statistical analysis (using SAS algorithms)
Inc. 1987) of data presented in Fig. 4.5.
(SAS Institute
General Linear Models Procedure
Class Level Information
Levels
Class
Values
ABA
4
ABO AB2 AB3 AB5
IRRAD
3
D FR R
Number of observations in data set = 146
General Linear Models Procedure
Dependent Variable: GERM
Sum of
DF
Squares
Source
11
87403.63194
Model
134
40718.94946
Error
128122.58140
Corr. Tot 145
R-Square
0.682188
Mean
Square
F Value
7945.78472
26.15
303.87276
C.V.
36.48367
Root MSE
17.43195
Pr > F
0.0001
GERM Mean
47.7801370
General Linear Models Procedure
Dependent Variable: GERM
Source
ABA
DF
3
IRRAD
2
ABA*IRRAD
6
Source
ABA
DF
IRRAD
2
ABA*IRRAD
6
3
Type I SS
31586.63887
53323.22322
2493.76984
Mean Square
10528.87962
26661.61161
415.62831
F Value
34.65
87.74
1.37
Pr > F
0.0001
0.0001
0.2321
Type III SS
32623.79719
49804.43903
2493.76984
Mean Square
10874.59906
24902.21952
415.62831
F Value
35.79
81.95
1.37
Pr > F
0.0001
0.0001
0.2321
157
General Linear Models Procedure
Least Squares Means
ABA
GERM
LSMEAN
Std Err
LSMEAN
37.0666667
71.3084615
85.8294118
29.0833333
40.8827273
74.4230769
19.2454545
36.5600000
68.8833333
7.1745455
21.4591667
46.7833333
4.5009092
4.8347521
4.2278680
7.1165623
5.2559296
4.8347521
5.2559296
5.5124655
5.0321695
5.2559296
5.0321695
4.5009092
IRRAD
ABO
ABO
ABO
AB2
AB2
AB2
AB3
AB3
AB3
AB5
AB5
AB5
D
FR
R
D
FR
R
D
FR
R
D
FR
R
Pr >
LSMEAN
Number
ITS
HO:LSMEAN=0
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0004
0.0001
0.0001
0.1745
0.0001
0.0001
1
2
3
4
5
6
7
8
9
10
11
12
Least Squares Means for effect ABA*IRRAD
Pr > Ti HO: LSMEAN(i)=LSMEAN(j)
I
Dependent Variable: GERM
2
1
i/i
0.0001
0.0001
0.0001
4
0.3448
5
0.5822
6
0.0001
7
0.0111
8
0.9433
9
0.0001
10 0.0001
11 0.0223
12 0.1292
0.0254
0.0001
0.0001
0.6495
0.0001
0.0001
0.7287
0.0001
0.0001
0.0003
2
3
4
3
.
1
.
0.0001
0.0254
.
0.0001
0.0001
0.0780
0.0001
0.0001
0.0110
0.0001
0.0001
0.0001
5
0.3448
0.0001
0.0001
.
0.1846
0.0001
0.2681
0.4077
0.0001
0.0145
0.3833
0.0374
0.5822
0.0001
0.0001
0.1846
.
0.0001
0.0042
0.5713
0.0002
0.0001
0.0085
0.3953
6
0.0001
0.6495
0.0780
0.0001
0.0001
0.0001
0.0001
0.4287
0.0001
0.0001
0.0001
7
0.0111
0.0001
0.0001
0.2681
0.0042
0.0001
0.0246
0.0001
0.1067
0.7614
0.0001
8
0.9433
0.0001
0.0001
0.4077
0.5713
0.0001
0.0246
.
0.0001
0.0002
0.0450
0.1532
Dependent Variable: GERM
i/j
1
2
3
4
5
6
7
8
9
10
11
12
10
11
12
0.0001
0.0001
0.0001
0.0145
0.0001
0.0001
0.1067
0.0002
0.0001
0.0223
0.0001
0.0001
0.3833
0.0085
0.0001
0.7614
0.0450
0.0001
0.0517
0.1292
0.0003
0.0001
0.0374
0.3953
0.0001
0.0001
0.1532
0.0014
0.0001
0.0003
9
0.0001
0.7287
0.0110
0.0001
0.0002
0.4287
0.0001
0.0001
.
0.0001
0.0014 0.0517
0.0001 0.0003
.
.
.
NOTE: To ensure overall protection level, only probabilities associated
with pre-planned comparisons should be used.
158
NOTE: The interaction between ABA and irradiation (ABA*IRRAD) was
significant only at the 0.23 level. Pre-planned comparisons (see "Least
Square Means" Tables) showed a clear promotion of FR-induced germination
above the dark control when no ABA was added to the incubation medium.
With addition of ABA, the promotive effect of FR irradiation was of
reduced magnitude, and sometimes FR was not significantly different
(P>0.05) from the dark control (e.g. ABA 0.02 and 0.05 mM).