AN ABSTRACT OF THE THESIS OF
Yerko M. Moreno-Simunovic for the degree of Doctor of Philosophy in
Horticulture presented on July 16, 1993.
Title: CALORIMETRIC STUDY OF THE RESPIRATORY METABOLISM OF
PEACH (Prunus oersica L. Batsch.) SEEDS DURING STRATIFICATION AND
ITS RELATIONSHIP TO SEEBjJNG GROWTH
/-)
Abstract approved:
=
Anita N. Azarenko
The respiratory metabolism of 'Lovell' and 'Nemaguard' peach seeds
during stratification, and their seedlings, was studied. The heat of
metabolism (qmet), COj evolution were measured in the isothermal mode of
a differential scanning calorimeter (DSC). qmet and COj increased with
stratification time for both cultivars. The ratio qmet/C02 evolution, an index
of metabolic efficiency, was calculated. A high ratio represents an
inefficiency of metabolism where a high proportion of the energy produced
by the tissue is lost as heat and not used for biomass production. For
'Lovell' seeds, metabolic efficiency was low in unstratified and partially
stratified seeds, but increased after 6 weeks of stratification. The time
required for germination was positively correlated with the qmet/C02 ratio
whereas germination percentage was negatively correlated. For
'Nemaguard', there was no correlation between the qmet/C02 ratio and
percent or number of days to germination. The temperature coefficient of
metabolism (//) of peach seeds was determined by DSC. As temperature
increased, fj decreased, depending on cultivar and stratification treatment.
Increasing stratification time produced taller seedlings for both cultivars
when measured 4 weeks after planting. C02 evolution increased and qmet
of seedling terminal shoots decreased with stratification time. The qmet/C02
ratio of 'Lovell' seedlings decreased with stratification treatment and was
inversely correlated to seedling growth, implying a more efficient
metabolism as the duration of stratification increased. This correlation was
not significant for 'Nemaguard'. The cytochrome (CP) and alternative (AP)
respiratory pathway capacities in stratifying embryos of 'Lovell' were also
measured. Respiratory inhibitor studies showed a significant AP capacity
present during the entire stratification treatment. CP capacity increased
steadily during stratification and became significantly higher than AP
capacity after 6 weeks of stratification. This change in capacities in seeds
was significantly correlated to the increase in germination percentage and
seedling height. GA3 applications after stratification stimulated an increase
qmet and C02 evolution rates of embryos during the first 4 weeks of
stratification, reducing the qmet/C02 evolution ratio to levels comparable to
those of fully stratified seeds. Even though GA3 increased respiratory
efficiency and stimulated germination, its effects on seedling growth did
not completely substitute for the lack of chilling for unstratified and
partially stratified seeds.
CALORIMETRIC STUDY OF THE RESPIRATORY METABOLISM OF PEACH
(Prunus persica L. Batsch.) SEEDS DURING STRATIFICATION AND ITS
RELATIONSHIP TO SEEDLING GROWTH
By
Yerko M. Moreno-Simunovic
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed July 16, 1993
Commencement June 1994
APPROVED:
^
1
TT
Professor of Horticulture in charge of major
—^
^j
Head of the Department of Horticulture
Dean of the G/^dqiate School/
Date thesis is presented July 16, 1993.
Typed by Yerko M. Moreno-Simunovic
Note:
This thesis is presented in manuscript format as three
publications written in the format of the Journal of the American
Society for Horticultural Science.
This thesis is dedicated to my parents. Carmen and Jose, my wife Cuty
and my son Jose Vicente. All of you have my love forever.
ACKNOWLEDGEMENTS
I wish to express my profound gratitude to all who so generously
contributed to the development and completition of this research project.
My major professor. Dr. Anita Azarenko, has been and excellent
advisor. Without her creative enthusiasm, understanding and support I would
have never been able to complete my degree.
I thank her for always
encouraging me to do my best and for showing me that no matter what, we
are human beings before scientists. More than my professor she is my friend
and will always be.
I also appreciate the useful input and advice provided by my committee
members, Drs. Pat Breen, Mike Penner, Mike Burke and my graduate
representative Dr. Alvin Mosley. Each of you was fundamental on a particular
phase of my studies and I thank you for that.
I would also like to thanks my sponsors MIDEPLAN-Chile and
Universidad de Talca and for providing partial financial support for my studies.
Special thanks goes to all the people I have had the opportunity to work
with. We will always share good memories and beautiful friendships. I would
like to mention my graduate student buddies Habib, Cheryl, "the two
Claudias", Pilar, Juan Pablo, Kirk, Alfonso, Glen, Becky, Ruth, Jorge, Enrique,
Anne, Neil, Kaiz, Carlos and Ana.
Drs. Daryl Richardson, Tim Righetti and
Porter Lombard have been and will continue to be very good friends. Many
thanks go to my good friends Ricardo and Anita, Rodrigo, Chino, Waleska,
Claudio and Patricia, Ricardo, Mauricio, Erika and many more that I cannot
include here due to space limitations.
My deepest gratitude and love goes to my wife 'Cuty' for her
encouragement, friendship and love.
friend and for that I love you.
More than my wife you are my best
TABLE OF CONTENTS
CHAPTER 1. INTRODUCTION
1
CHAPTER 2. LITERATURE REVIEW
5
Seed Dormancy
5
Plant Respiration
17
Differential Scanning Calorimetry (DSC)
30
CHAPTER 3. METABOLIC ACTIVITIES OF PEACH SEEDS DURING
DORMANCY REMOVAL AND EARLY SEEDLING GROWTH .... 37
Abstract
37
Introduction
39
Materials and Methods
42
Results and Discussion
46
Literature Cited
63
CHAPTER 4. CHANGES IN THE CAPACITIES OF THE CYTOCHROME
AND ALTERNATIVE RESPIRATORY PATHWAYS MEASURED BY
DIFFERENTIAL SCANNING CALORIMETRY DURING THE
STRATIFICATION OF PEACH SEEDS
66
Abstract
66
Introduction
67
Materials and Methods
70
Results
73
Discussion
76
Literature Cited
84
TABLE OF CONTENTS, CONTINUED
CHAPTER 5. METABOLIC EFFICIENCY OF STRATITYING PEACH
SEEDS IS AFFECTED BY GIBBERELLIN TREATMENTS
88
Abstract
88
Introduction
89
Materials and Methods
91
Results
95
Discussion
97
Literature Cited
103
BIBLIOGRAPHY
106
APPENDICES
125
LIST OF FIGURES
Figure 3.1.
Figure 3.2.
Figure 3.3.
Figure 3.4.
Figure 3.5.
Figure 3.6.
Figure 3.7.
Figure 4.1.
Days to germination and percentage of germination (A)
and (B) changes in heat of metabolism (qme,), C02
evolution rates and in the qmet/C02 evolution ratio of
'Lovell' peach seeds stratified at 4C for 0 to 12 weeks. .
56
Days to germination and percentage of germination (A)
and (B) changes in heat of metabolism (qmet), CO2
evolution rates and in the qmet/C02 evolution ratio of
'Nemaguard' peach seeds stratified at 4C for 0 to 7
weeks
57
Differential scanning calorimetric determination of the
metabolic heat rates of 'Lovell' peach seeds stratified for
0, 2, 4, 6, 8, 10, and 12 weeks at 4C. Direct calorimetric
data (A) and Arrhenius plots (B)
58
Differential scanning calorimetric determination of the
metabolic heat rates of 'Nemaguard' peach seeds stratified
for 0, 1, 2, 3, 4, 5, 6, and 7 weeks at 4C. Direct
calorimetric data (A) and Arrhenius plots (B)
59
Temperature coefficients of metabolism (//) of peach seeds
cv. Lovell stratified for 0, 2, 4, 6, 8, 10, and 12 weeks at
4C (A) and average temperature coefficient of
metabolism for 'Nemaguard' seeds stratified for 0 to 7
weeks (B)
60
Seedling height (A) and (B) changes in heat of metabolism
(qmet), CO2 evolution rates and in the qmet/C02 evolution
ratio for seedlings obtained from seeds of cv. Lovell
stratified at 4C for 2 to 12 weeks
61
Seedling height (A) and (B) changes in heat of metabolism
(qmet), C02 evolution rates and in the qmet/C02 evolution
ratio for seedlings obtained from seeds of cv. Nemaguard
stratified at 4C for 2 to 7 weeks
62
Effect of sodium azide (NaNg) concentrations in the
absence (A) or presence (B) of 10"3M SHAM on the rate of
heat evolution at 24C of peach seeds stratified at 4C for
0 to 12 weeks.
Each line intersection of the grid
represents a mean of three replicates
81
LIST OF FIGURES, CONTINUED
Figure 4.2. Effect of salycylhydroxamic acid (SHAM) concentrations
in the absence (A) or presence (B) of 10"3M sodium azide
on the rate of heat evolution at 24C of peach seeds
stratified at 4C for 0 to 12 weeks. Each line intersection
of the grid represents a mean of three replicates
82
Figure 4.3. Cytochrome and alternative pathway respiratory capacity
of peach seeds stratified at 4C for 0 to 12 weeks. Each
point represents a mean of three replicates ±SE
83
Figure 5.1. Germination percentages (A) and seedling height (B) for
peach seeds cv. Lovell treated with 0 or 500 mg • I1 GA3,
after stratification at 4C for 0 to 12 weeks
100
Figure 5.2.
Figure 5.3.
Changes in heat of metabolism (qmet) (A), C02 evolution
rates (B) and in the qmet/C02 evolution ratio (C) of 'Lovell'
peach cotyledons treated with with 0 or 500 mg • I'1 GA3,
after stratification at 4C for 0 to 12 weeks
101
Changes in heat of metabolism (qmet) (A), C02 evolution
rates (B) and in the qmet/C02 evolution ratio (C) of 'Lovell'
peach embryos treated with with 0 or 500 mg • I"1 GA3,
after stratification at 4C for 0 to 12 weeks
102
LIST OF TABLES
Table 3.1.
Table 4.1.
Maximum heat evolution and break temperatures from
temperature scanning experiments with peach seeds of
cuitivars Lovell and Nemaguard, stratified at 4C for 0 to
12 and 0 to 7 weeks respectively2
55
Correlations of the mean cytochrome and alternative
pathway activities and the number of days to germination,
germination percentage and seedling height for peach
seeds cv. Lovell, stratified for 0 to 12 weeks
80
LIST OF APPENDIX TABLES
Appendix 1.
Appendix 2.
Appendix 3.
Appendix 4.
Analysis of variance for the temperature coefficient
of metabolism (//) of 'Lovell' seeds stratified at 4C
for 0 to 12 weeks
125
Analysis of variance for the temperature coefficient
of metabolism (//) of 'Nemaguard' seeds stratified
at 4C for 0 to 7 weeks
126
Analysis of variance for the qmet and C02 evolution
rates and the qmet/C02 ratio for 'Lovell' cotyledons
stratified at 4C for 0 to 12 weeks and treated with
0 or 500 ppp GAg
127
Analysis of variance for the qimet
and CO, evolution
n
rates and the qmett/COj
/
ratio for 'Lovell' embryos
stratified at 4C for 0 to 12 weeks and treated with
0 or 500 ppp GA3
128
CALORIMETRIC STUDY OF THE RESPIRATORY METABOLISM OF PEACH
(Prunus persica L. Batsch) SEEDS DURING STRATIFICATION AND ITS
RELATIONSHIP TO SEEDLING GROWTH
CHAPTER 1
INTRODUCTION
The seeds of peach and many other Rosaceous species require a period
of stratification (e.g. incubation in moist medium at cold temperatures) before
they will germinate and develop normally. If the chilling requirements of these
seeds are not fully satisfied, they may germinate but the resultant seedlings
are physiological dwarfs (Davidson, 1934; Flemion, 1934; Tukey and Carlson,
1945; Pollock, 1962; Flemion and Beardow, 1963; Zigas and Coombe, 1977;
Frisby and Seeley, 1993a,b). The germination of the seeds and the growth
capacity of the resulting seedlings increase with
longer stratification
treatments, but the relationship between seed germination (or the cessation
of dormancy) and seedling growth is not well understood.
Stratification treatments have been shown to produce a large increase
in respiratory activity (Pollock and Olney, 1959; Pollock, 1960; La Croix and
Jasval, 1967).
No reports are available relating respiratory changes during
seed stratification with the metabolic activity of resultant seedlings and
seedling vigor. Most of the reports regarding the respiratory metabolism of
dormant seeds have focused on the activities of glycolysis and pentose
phosphate pathways during stratification (La Croix and Jasval, 1967; Bogatek
2
and Lewak, 1988; Hu and Couvillon, 1990). According to other reports, the
alternative (cyanide-resistant) respiratory pathway may play a role in
respiratory metabolism during dormancy termination and germination of seeds
and thus influence seedling growth (Yentur and Leopold, 1976; Burguillo and
Nicolas, 1977; James and Spencer, 1979; Esashietal., 1981, 1982; Adkins
et al. 1984; Stewart et al., 1990b).
The seeds of many plant species,
including Prunus spp. contain cyanogenic glucosides that upon the action of
fc-glucosidase liberate hydrogen cyanide (Esashi et al., 1991a).
The HCN
liberated may inhibit the operation of the cytochrome pathway of respiration
and, as a result, increase the flux of electrons through the CN-resistant
pathway. However, Alscher-Herman et al., (1981) and Bogatek and Rychter
(1984) investigated the respiratory activity of pear and apple seeds during
stratification and found a progressive increase in respiration that, according
to their results, did not involve an important contribution of the alternative
pathway of respiration.
Several treatments have been used to overcome seed dormancy.
Among them, gibberellins eliminated the chilling requirement for peach and
apple seeds, increasing germination percentage and in some cases stimulating
seedling growth (Zigas and Coombe, 1977b; Rouskas et al., 1980; Mehanna
et al., 1985). Not many reports relating GA treatments to respiratory activity
are available but one study showed an effect of GA3 in altering the respiratory
3
pathways during early seed germination and stimulating alternative respiratory
activity (Gui et al., 1991).
The measurement of heat evolution from plants tissues constitutes a
sensitive, non-invasive way to measure their rates of metabolic activity (Loike
et al., 1981), and provides a way to estimate the relative efficiency of plant
metabolism at a particular stage of development or under a given set of
environmental conditions (Criddle et al., 1990).
Recently, Gardea et al.
(1993) characterized metabolic changes during the dormancy development of
primary grape buds. They used the temperature coefficient of metabolism {JJ)
and the ratio qmet/C02 evolution to study changes in the efficiency of plant
metabolism during this period.
Prediction of the long term growth of larch
clones by calorimetric measurements of metabolic heat rates has been studied
(Hansen et al., 1989).
I have hypothesized that the stratification treatment of peach seeds
alters their respiratory metabolism which, when the chilling treatment is
complete, results in the maximum percentage of germination and seedling
vigor. Differential temperature-scanning and isothermal microcalorimetry was
used to characterize changes in the respiratory metabolism of peach seeds
during stratification and the possible relationship of these changes to seedling
growth. One of the many potential effects that stratification may have is on
the form of respiration (e.g. cytochrome or alternative pathways) and/or the
changes that occur in their capacities during the stratification process. The
4
alternative pathway produce less energy in the form of ATP and more heat
thereby resulting in a less efficient metabolism.
Therefore, the possibility
exists that as the stratification duration increases, the proportion of respiratory
metabolism that is contributed by the alternative pathway becomes less and
the cytochrome oxidase system contributes more. This change could result
in the peach seeds having a more efficient metabolism because of more ATP
production, less energy lost as heat and better substrate utilization.
This
increase in metabolic efficiency may result in the maximum germination
potential and a more vigorous phenotype. The research also focussed on
testing this hypothesis.
Lastly, gibberellins have been shown to at least
partially compensate for the chilling requirement in peach seeds. I determined
the effect of GA3 on the metabolic efficiency of stratifying peach seeds.
Again if metabolic efficiency is increased by GA3 then germination and growth
potential may be maximized.
This information could lead to the further
development of a model which incorporates a direct or indirect role of this
plant hormone in respiratory metabolism.
CHAPTER 2
LITERATURE REVIEW
Seed Dormancy
Definitions and Terminology
Attempts to discuss the various aspects of plant dormancy can be
bewildering due to the excessive number of non-physiological independent
terms that have arisen over the years (Lang et al., 1987). Some of the terms
used today are those proposed by Doorenbos (1953) who indicated that
plants go through periods of growth by elongation, alternating with periods in
which no such growth occurs (Lang et al., 1987). This state of inactivity was
called 'dormancy'.
Doorenbos review proposed the use of three terms:
'imposed dormancy' when the environment imposes growth inactivity,
'summer dormancy', when terminal buds inhibit lateral buds, and 'winter
dormancy' when the cause of dormancy is not systemic, but lies within the
tissue itself.
Another group of terms was independently proposed by Samish (1 954),
who defined 'dormancy' as the temporary suspension of any visible growth,
especially of buds and seeds, despite its cause. Quiescence was defined as
that state in which external conditions stopped growth. Those cases where
6
internal factors caused the dormant state even under favorable conditions,
were called 'rest'.
Other researchers have used more than
50 different terms or
modification of the above ones, many with duplicate meanings. Lang et al.
(1987) attempted to simplify the terminology proposing "a descriptive system
of physiological terminology, based on processes and inputs that can be easily
understood, translated, and expanded as complexities are revealed". They
coined three terms to classify dormancy.
'Endodormancy' implies that the
initial reaction leading to growth cessation is a specific perception of an
environmental or endogenous signal within the affected structure alone.
'Paradormancy' involves a specific biochemical signal originating in a structure
other than the affected structure as an initial reaction.
'Ecodormancy'
includes all cases of dormancy resulting from one or more unsuitable
environmental factors, which are generally nonspecific in their effect on
overall plant metabolism. This last series of terms will be used on this review.
Changes on Nucleic Acids or the Genetic Control of Dormancy
The genetic control of development has probably been the most difficult
aspect of contemporary biology to investigate (Ross, 1985). The idea that
certain genes are expressed only during seed development, maturation or
germination is not usually doubted. However, the well known hypothesis of
Tuan and Bonner (1964) that plant organs are dormant because of direct
7
repression of some part of the genome remains to
demonstrated.
be conclusively
For dormancy to be overcome in certain species, including
Norway maple {Acer platanoides L), hazelnut {Cory/us avellana L), peach
(Prunus persica L. Batsch) and others, the seed must undergo a period of
exposure to low temperatures under moist conditions (stratification). Slater
and Bryant (1982) noticed that the stratified seed of Norway maple
accumulated RNA in the embryonic axis.
The synthesis of RNA was
measured by the incorporation of radiolabelled precursors and most of the
newly synthesized RNA was ribosomal RNA (rRNA). An earlier investigation
by Wood and Bradbeer (1967) using hazelnut seeds had yielded similar
results. In their experiments other forms of RNA were also labelled and there
were no significant differences between stratified and non-stratified seeds in
relation to the proportion of the [3H]uridine incorporation associated with poly
(A)-rich RNA.
For this reason it was postulated that the unmasking of the
genome did not occur, and the increasing growth potential brought about by
chilling was attributed to an increase in the protein-synthesizing machinery,
mainly RRNA (Slater and Bryant, 1982).
Other reports on nucleic acid metabolism following gibberellin (GA3)
treatment do show differences in nucleic acid labelling (Jarvis et al., 1968;
Jarvis and Shannon, 1981).
Nevertheless according to Ross (1985) it is
necessary to recognize that since treatment of seeds with GA normally
stimulates germination and its associated different and increased metabolic
8
activities, this experimental approach cannot yield any information on the
natural means of breaking dormancy.
While following metabolic changes during the dormancy period of
cherry flower buds Wang et al. (1985) reported that a sharp increase in DNA
and RNA content characterized the transition from dormancy to active growth
on late and early blooming cultivars. Macdonald and Osborne (1988) reported
that continuous synthesis of protein, RNA and DNA occurred in potato tuber
buds from the time of tuber harvest through dormancy. According to their
findings breaking of dormancy is associated with an increase in all cellular
activities. Other researchers have considered the limitations of determining
only quantitative changes of nucleic acids and proteins, pointing out the need
for determining changes of specific mRNA species and their associated
transcripts (Hofmann and Hand, 1992; Dommes, 1988).
In one of these
studies, polyadenylated RNA was translated from dormant potato tubers at
different times during dormancy and translated in vitro.
No changes were
noted in protein patterns but many differences occurred in translatable mRNA.
Among 300 polypeptides resolved, 5rshowed a dramatic decrease, 7
increased, indicating that release from dormancy was accompanied by
changes in gene expression (Dommes, 1988).
Studies by Goldmark et al. (1992) and Morris et al. (1991), showed
differences in mRNA species between hydrated dormant and non-dormant
seeds of cereals. A cDNA clone, pBS128, was identified in Bromus secalis.
9
This clone encodes a transcript that upon imbibition increases over four fold
in dormant seeds but rapidly declines and disappears in non-dormant seeds.
Levels of this embryo specific transcript were enhanced by 50 /vM ABA but
the putative protein sequence shows no resemblance to other reported ABA
responsive genes.
Metabolic Changes During Dormancy
Hormonal changes. The concept of a balance of promoter-inhibitor was
first applied to seeds by Villiers and Wareing (1960) in their study of
dormancy in ash (Fraxinus excelsior L.) and later elaborated by Amen (1968)
and Khan (1971, 1975).
The model predicts a change in the hormonal
balance in response to environmental factors, which would then trigger the
activation of metabolism that leads to germination. This means that inhibitory
substances must predominate in the dormant seeds, then in response to an
environmental signal, promoter hormones are synthesized, and germination
results. Experimentation has not provided evidence that confirms the validity
of this model (Ross, 1985). Trewavas (1981) makes the point that many of
the assumptions are not founded and that many of the classical experimental
data do not support the original hypothesis.
The most consistent evidence for the involvement of growth regulators
in seed dormancy comes from studies on ABA. It has been shown that many
maturing seeds accumulate ABA (King, 1976), even in those species that do
10
not exhibit a true physiological dormancy like pea (Pisum sativum L).
Changes in ABA content have been studied in seeds and buds of several
species to determine if a relationship exists between ABA and changes in
dormancy during chilling (Barthe and Bulard, 1978; Borkowska and Powell,
1982; Bowen and Dickerson, 1978; Diaz and Martin, 1972; Halinska and
Lewak, 1978; Mielke and Dennis, 1975, 1978; Orlando and Dennis, 1977;
Rudnicki, 1969; Sharma and Singh, 1980).
Many of these studies show a
decline in ABA during chilling. The conclusion in these cases is that chilling
temperatures are responsible for the decline in ABA, and that this decline is
important for the reduction of dormancy intensity (Powell, 1987). The chilling
process appears to lead to the production of some type of growth-promoting
force or substance (e.g. gibberellins) (Powell, 1987). Rudnicki (1969) found
that much of the ABA in apple seeds disappeared after 3 weeks of
stratification and that only limited germination was possible at this time. As
stratification proceeded, germination continued to improve.
Increasing
amounts of exogenous ABA were required to block the germination process
as stratification proceeded, indicating that a growth promoting substance was
being generated.
Whether this force was hormonal in nature, or reflected
changes in some other type of cellular activity was not determined. So at this
point it seems clear that ABA may play an important role in early seed and
bud dormancy but not in regulating growth at later stages (Powell, 1987).
11
Evidence for possible involvement of gibberellins (GA) in dormancy
comes partially from exogenous applications of these compounds. In hazelnut
seeds,
several inhibitors that interfered with
GA production (e.g.
2-
chloroethyltrimethyl ammonium chloride), also interfered with germination of
chilled seeds (Ross and Bradbeer, 1971).
Exogenous GA applications have
partially or totally replaced the chilling requirement of hazelnut seeds (Jarvis
et al., 1968) and that of peach seeds (Donoho and Walker, 1957; Gianfagna
and Rachmiel, 1986).
Endogenous gibberellins have also been studied extensively with
respect to their role in the chill-related dormancy mechanism of seeds
(Gianfagna and Rachmiel, 1983, 1986; Isaia and Bulard, 1978; Ross and
Bradbeer, 1971).
In general, the amount of gibberellins is small in dormant
seeds but increases during the chilling process. Stratified hazelnut seeds must
be placed in warm germinating temperatures before a substantial amount of
gibberellin is detectable, suggesting that the chilling process affects the
gibberellin biosynthetic capacity of the plant (Ross and Bradbeer, 1971).
Bianco et al. (1984) found large quantities of GA in dormant embryos of
'Golden Delicious' apples extracted with Tris buffer, in contrast to trace
amounts extractable with conventional ethanolic solvents, suggesting that
chilling temperatures may affect GA biosynthetic capacity of the seeds
through an effect on membrane permeability. In relation to the probable mode
of action of gibberellins, Zarska-Maciejewskaetal. (1980) found evidence that
12
gibberellins may act through accumulation of acid lipase, although they noted
that gibberellin-mediated hydrolysis of reserve lipids can hardly be the only
mechanism involved in the removal of embryo dormancy in apple.
In a
complementary work, Ross (1983) also noted the presence of a lipase in
hazelnut seeds with a temperature optimum of 50C.
The evidence for the involvement of cytokinins in the release of organs
from dormancy is not as extensively documented as for ABA and gibberellins.
Rest-breaking treatments cause a rapid increase in xylem sap cytokinin
concentration of apple trees, peaking just before or at budbreak (Cutting et al.,
1991).
The same response was found in extracts of breaking buds from
sugar maple (Taylor and Dumbroff, 1975).
Application of cytokinins to
dormant or partially chilled seeds has promoted growth in some cases, but not
in others (Lewak and Bryzek, 1974; Rudnicki et al., 1973; Tzou et al., 1973).
There is little evidence that auxins play an important role in regulating
rest in buds or chill-requiring seeds (Powell, 1987). Several reports show that
IAA in seeds of Pinus silvestris L, Acer platanoides L, and Rosa rugosa
Thunb. has no regulatory role in seed dormancy (Tillberg, 1977, 1984; Tillberg
and Pinfield, 1981).
Many studies indicate that ethylene can stimulate the germination of
seeds and growth of buds, but most of these investigations do not show
conclusive evidence that ethylene plays an important regulatory role in the
dormancy mechanism in seeds and buds for which chilling is required (Powell,
13
1987).
Even in those studies that found a positive response, this was
generally attributed to ethylene action on events after partial or full release
from dormancy by chilling (Kepczynski et al., 1977; Paiva and Robitaille,
1978; Wareing, 1956; Zimmerman et al., 1977).
A report by Sinska (1989) showed that germination of dormant and
partly stratified apple embryos was enhanced by ethephon, benzylaminopurine
(BA) and gibberellin and that an additional increase in germination was
observed when growth regulators were used in combination. The effect of all
three growth regulators was greatest for dormant and briefly stratified
embryos, and diminished with stratification.
BA and GA3 counteracted the
inhibitory effect of aminooxyacetic acid (AOA) and aminoethoxyvinylglycine
(AVG) on embryo germination; the effect of BA was comparable to the effect
of ethephon, while the effect of GA3 was greater than that of ethephon.
These results and others by the same author suggest that BA could play a role
in dormancy regulation through a direct or indirect action on ethylene
production, counteracting the effect of high levels of ABA that participate in
dormancy maintenance in this species. Finally, this report shows again that
the dormancy phenomenon is not as simple as originally thought and that
growth regulators are only some of the factors affecting its control.
Mobilization of reserve materials and other changes.
The metabolic
processes that occur during stratification of seeds from several species have
been investigated extensively but no conclusive evidence about their role in
14
the removal of dormancy and seedling growth has been found.
In apple
seeds, the role attributed to the hydrolysis of reserve materials has received
particular emphasis (Bouvier-Durand et al., 1984). In dormant seeds of this
species, storage proteins and fats are the main reserves, with starch being
less abundant (Davidowicz-Grzegorzewska and Lewak, 1978; Bouvier-Durand
et al., 1981).
Lewak et al. (1975) postulated that protein hydrolysis is an
essential step in the removal of embryo dormancy.
Similarly, changes in
starch content (Davidowicz-Grsegorzewska and Lewak, 1978) and hydrolysis
of reserve lipids (Zarska-Maciejewska and Lewak, 1976) were considered to
be involved in the same process. At the same time, numerous data on various
seeds, including apple, indicated that reserve mobilization does not seem to
be involved in dormancy breaking (Bewley and Black, 1982; Bouvier-Durand
et al., 1984).
Whether or not these processes are involved in subsequent
seedling growth and the expression of a normal phenotype has not been
determined.
The seeds of many species, including non-cyanogenic plants, evolve
HCN gas during pre-germination (Esashi et al., 1991).
HCN is supposedly
liberated from bound cyanogenic compounds present as storage substances
within seeds. The cyanogenic reserves in the seeds of cyanogenic species are
usually cyanohydrins, cyanogenic glucosides (Wokes and Wilhimont, 1951;
Dziewanowska et al.,
(Mikolajczak, 1977).
1979a,b;
Seigler,
1981)
and cyanogenic lipids
Such compounds are also present in seeds of non-
15
cyanogenic species (Esashi et al., 1991 a). If the release of HCN by seeds is
present in all kinds of plant species, including non-cyanogenic ones, the HCN
liberated may directly inhibit the operation of the cytochrome pathway and,as
a result, increase the flux of electrons through the cyanide-resistant pathway
(Esashi et al., 1991b).
Seedling Growth and Endodormancy
It has been shown that embryos excised from non-stratified seeds will
germinate, but the rate and the extent of germination and the nature of the
seedling which is formed reflect the physiological state of the seeds from
which the embryos were taken (Bewley and Black, 1982).
Seedlings from
seeds or excised embryos that have received a minimal stratification treatment
usually grow abnormally (Alscher-Herman and Khan, 1980; Come, 1970;
Chao and Walker, 1966; Flemion, 1936; Flemion and De Silva, 1960; Pollock,
1962; Tukey and Carlson, 1945).
Full and 'normal' germination of excised
apple embryos is reached sometime before the seeds are fully stratified
(before intact seeds will germinate) (Maciejewska et al., 1974).
Peach seeds require a stratification treatment varying from 6 to 14
weeks to germinate and produce normal seedlings (Westwood, 1993).
Several workers have been able to germinate seeds without chilling by
removal (Tukey, 1933; Flemion, 1934; Lammerts, 1942; Mehanna and
Martin, 1985) or puncturing of the seed coat (Chao and Walker, 1966). Yet
16
in most of these investigations, resultant seedlings were stunted, and showed
leaf lesions and poor root development (Tukey, 1933; Flemion, 1934;
Lammerts, 1942; Chao and Walker, 1966; Zigas and Coombe, 1977a). This
abnormal seedling growth is called physiological dwarfing and is characterized
by small size, especially of the primary stem, abnormal (epinastic) leaf
development on the primary stem, and abnormal leaf development in lateral
branches (Flemion, 1959; Pollock, 1962).
Occasionally, severely dwarfed
seedlings appear to begin normal growth, but this is due to growth of a lateral
branch originating near the terminal end of the primary stem (Davidson, 1934;
Tukey and Carlson, 1945; Wang and Beardow, 1968). According to Flemion
and Waterbury (1945) physiological dwarfing of peach seedlings does not
prevent normal root growth and development.
However other researchers
have shown that peach seedlings arising from chilled embryos with intact seed
coats were taller and had a greater dry weight of shoots, leaves and roots
than seedlings arising from non-chilled embryos without seed coats (du Toit
etal., 1979).
Abnormal leaf development has been used as an
physiological dwarfing (Flemion,
1959; Pollock, 1959;
indicator of
Pollock, 1962).
However Wang and Beardow (1968) have suggested that physiological
dwarfing and abnormal leaf development are two separate growth problems.
They characterized physiological dwarfing by the small size and short
internodes of seedlings, and determined that a shorter period of stratification
17
is required to overcome true dwarfing than is required to overcome abnormal
leaf development.
Their conclusion was that apex abortion coupled with
abnormal leaves causes a plant to appear dwarfed when it would otherwise
have grown normally.
The relationship between endodormancy and seedling growth has not
been extensively studied.
Chandler et al. (1937) suggested that delayed
foliation and physiological dwarfing are manifestations of the same problem,
consistent with the fact that seed stratification enhances germination and
reduces abnormal seedling growth.
Frisby and Seeley (1993a) studied this
relationship only to find the same confounding effects between true
physiological dwarfing and abnormal leaf development.
Plant Respiration
Definition and Stoichiometry
Respiration is an oxidative process in which complex molecules such as
carbohydrates and fats are broken down into C02 and H20 and a high
proportion of the liberated free energy is conserved as ATP (Goodwin and
Mercer, 1988).
The overall process is complex and can be divided into a number of
reaction sequences (e.g. glycolysis, tricarboxylic acid cycle, the oxidative
pentose phosphate pathway, the fatty acid B-oxidation spiral) (Stryer, 1988).
The function of most of these pathways is the generation of high-energy
18
reduced compound (e.g. NADH, succinate) at the expense of the substrates
being oxidized. These reductants are then re-oxidized by molecular oxygen
by means of the terminal electron transport chain, a sequence of redox
systems located in the inner membrane of the mitochondria. The movement
of the electron pair down a potential gradient (negative E'0 to positive E'0)
releases free energy that is conserved with an efficiency of about 40% by
using it to drive the endergonic phosphorylation of ADP as shown in the
following equation (Goodwin and Mercer, 1988)
ADP+H3P04~ATP+H20
DELTAG0'=+30.5kJmol-1
Based on this equation the only purpose of respiration is the generation
of ATP, which is often said to be the energy currency of the cell (Amthor,
1989). Other authors summarize the stoichiometry of respiration as:
C6Hn06+602+irttermediates~6C02+6HJ0+intermediates+6&6Kcal
In this equation sugars are the starting point and the result is usable
energy and metabolic intermediates, which are necessary for growth in
meristematic tissues, maintenance of existing phytomass, uptake of nutrients
and intra/intercellular transport of organic and inorganic materials (Amthor,
1989). As oxygen is needed to metabolize sugars (mainly glucose) and other
compounds, the result is a liberation of energy and with the formation of
water and evolution of COj. A total of 36 ATP's is formed from the oxidation
19
of a glucose molecule, assuming that the glycerol-phosphate shuttle is used
to transfer electrons from the cytosol to the respiratory chain in the
mitochondria.
This represents an investment of 263 kcal, out of the 686
liberated (Stryer, 1988). The remaining 423 kcal are dissipated as metabolic
heat.
According to this, all cellular metabolic activities are accompanied by
characteristic heat loss (Loike et al., 1981), and it can be said that heat
production is an inevitable byproduct of all growth processes (Criddle et al.,
1991).
Plant metabolic rates can be determined by three major methods
(Criddle et al., 1988). One is to determine biomass production over a given
time. The second method is to use some biochemical measure such as the
rate of C02 release, 02 uptake, or the incorporation of some labelled nutrient.
A third method of evaluating metabolism is to measure the heat evolution
rates, which are interpreted as a measurement of overall metabolic rate. This
will be discussed in detail later in the text.
Major Biochemical Pathways Involved in Respiration
G/yco/ysis and Pentose Phosphate Pathway (PPP). Glycolysis, and PPP,
both occur in the cytosol and use common substrates. Glucose 6-phosphate
isomerase and glyceraldehyde 3-phosphate dehydrogenase are the two most
important enzymes interconnecting these two pathways (Amthor, 1989).
20
PPP is a major route of glucose catabolism in several higher plants
(Humpreys and Dugger, 1957).
La Croix and Jaswal (1967) reported an
increasing activity of PPP, during the seventh week of stratification of cherry
seeds, parallel to a transition from endo- to ecodormancy. Zimmerman and
Faust (1969) concluded that overwintering pear buds respired mostly through
PPP. But during bud swelling, glucose metabolism changes with a decrease
in PPP activity, and a concomitant increase in the glycolytic pathway.
A
similar finding was reported by Bogatek and Lewak (1988) in apple seeds
during stratification.
Recent work (Perino and Come, 1991) described two
phases during germination of apple embryos, with the transition from
germination sensu stricto to growth being marked by changes in respiratory
activity.
Substances that inhibited respiration or that stimulated PPP, also
stimulated germination sensu stricto.
Mechanisms of Electron Transport
The immense complexity of the respiratory chain becomes apparent
when it is realized that a single respiratory chain unit (mol. wt. app. 1.52 x
106) contains as many as 40 redox centers and 50 polypeptides together with
a significant amount of phospholipids (Douce, 1985).
For many years, the organization of the respiratory chain of plant
mitochondria was thought to be very similar to that of animals, such as rat
liver or beef heart.
However there are a number of distinct differences
21
between plant and animal mitochondria; these include the cyanide-resistant
electron pathway, which is also encountered in the mitochondria of
microorganisms (Lloyd, 1974), and the rotenone-resistant electron pathway
(Douce, 1985).
Cytochrome oxidase pathway.
The plant mitochondrial respiratory
chain is responsible for the electron transfer from endogenous NADH and
succinate to 02 (Goodwin and Mercer, 1988). Both NADH and succinate are
formed in the mitochondrial matrix and initially oxidized on the inner surface
of the inner membrane. The chain consists of only five complexes: complex
I, complex II, complex III (called the cytochrome b-c, complex), cytochrome
c, and complex IV or cytochrome c oxidase. Except for cytochrome c, these
complexes are very hydrophobic and their fractionation procedure involves the
use of detergents as deoxycholate and cholate (Douce, 1985).
Complex I (NADH dehydrogenase) is that segment responsible for
electron transfer from NADH to ubiquinone and constitutes the entry point for
the redox equivalents of NADH produced in the mitochondrial matrix to the
respiratory chain. It contains a non-covalently bound flavin mononucleotide
(FMN), several iron-sulfur centers and two molecules of ubiquinone (Beinerts,
1977). The mitochondrial complex II (succinate dehydrogenase) is responsible
for electron transfer from succinate to ubiquinone (Hatefi and Siggall, 1976).
It is composed of two subcomplexes: succinate dehydrogenase and apo-Q
protein and contains FAD and several non-heme iron centers (Douce, 1985).
22
Complex III is responsible for electron transfer from ubiquinol (QH2) to
cytochrome c. It is composed of cytochromes b and c,, ubiquinones, and an
iron-sulfur center. Mitochondrial complex IV, also known as cytochrome c
oxidase is the terminal complex of the electron transport chain. It contains
two cytochromes (CuA and CuB). Cytochrome a reacts with cytochrome c,
while cytochrome 83 reacts with 02 and is accessible to other ligands (e.g. CN"
, N3" and CO) (Lemberg and Barret, 1973). This complex spans the membrane
asymmetrically and is responsible for the final transfer of electrons to oxygen
(Douce, 1985).
Cyanide resistant respiration. Cyanide-resistant respiration has been
reported in tissues from a large number of plants, including a variety of
angiosperms (mung bean, potato, pea, corn, wheat), fern mosses, algae and
various fungi (Henry and Nyns, 1975; Siedow and Girvin, 1980; Solomos,
1977). The classic and most striking example of cyanide-resistant respiration
occurs in the thermogenic spadix tissue of certain members of the family
Araceae (skunk cabbage, voodoo lily). In the spadices of such plants cyanide
resistant respiration contributes to respiratory rates similar to those found in
insect flight muscle (Siedow and Berthold, 1986). This cyanide-resistant 02
uptake takes place on the inner mitochondrial membrane and constitutes a
pathway alternative to the main cyanide-sensitive cytochrome pathway by
which electrons from reduced tricarboxylic acid (TCA) cycle substrates flow
to the terminal electron acceptor, Oj. Schonbaum et al. (1 971) showed that
23
the alternative pathway could be specifically inhibited by aryl-substituted
hydroxamic acids, of which salicilhydroxamic acid (SHAM) is the most widely
employed. This discovery led to the general characterization of the alternative
pathway as cyanide- resistant, hydroxamate-sensitive O2 uptake reaction
associated with plant mitochondria. Although much work has been devoted
to the pathway, the exact biochemical nature of the cyanide-resistant oxidase
is only beginning to be understood.
Following the initial characterization of the alternative oxidase as a
ubiquinol oxidase by Rich (1978), there have been several attempts to purify
this enzyme utilizing soluble ubiquinol analogs as substrates for the enzyme
(Huq and Palmer, 1978: Bonner and Rich, 1983; Elthon and Mclntosh, 1986,
1987).
In spite of these efforts, the alternative oxidase has proven to be
relatively difficult to purify. Elthon and Mclntosh (1987) partially purified an
alternative oxidase associated polypeptide on SDS-polyacrylamide gels when
working with tissues of the thermogenic spadix of Sauromatum guttatum.
Using polyclonal antibodies and a combination of cation-exchange and
hydrophobic interaction chromatography they were able to separate four
proteins with alternative oxidase activity. Their apparent molecular masses
were 37, 36, 35.5 and 35 kDa. The 37 kDa protein seems to be constitutive
in Sauromatum, whereas expression of the 36 and 35 kDa proteins was
correlated with the presence of alternative pathway activity. The 35.5 kDa
protein appears with the loss of alternative pathway activity, indicating that
24
it may be a degradation product of the 36 kDa protein (Elthon and Mclntosh,
1987). Elthon et al. (1989) later raised monoclonal antibodies against these
polypeptides, and the sequences of cDNA clones associated with these
proteins have all been reported (Rhoads and Mclntosh, 1991; Sakajo et al.,
1991). The alternative oxidase appears to be an integral membrane protein
and most attempts to purify it by solubilizing the mitochondrial membrane
with detergent treatment have resulted in a large loss of activity (Moore and
Siedow, 1991).
Recently
Berthold
and
Siedow
(1993),
using
the
detergent
deoxyBIGCHAP (/V,/\/-bis-(3-d-glucon-amido-propyl)deoxycholamide) and a
combination of chromatography and gel filtration techniques, were able to
obtain a 20- to 30 fold purification without contamination by cytochrome c
oxidase (complex IV) or cytochrome c reductase (complex III).
Their work
shows major polypeptides at 36 and 29 kDa, both of which react with
monoclonal antibodies raised against the Sauromatum guttatum protein by
Elthon etal. (1989).
Although the alternative pathway has been known for many years, little
progress has been made in elucidating its physiological role. Lyr and Schewe
(1975) suggested that the pathway represented a primitive electron transport
chain which was either lost or retained by different groups subsequent to
acquisition of the more efficient cytochrome pathway.
Palmer (1976)
proposed a more generalized physiological role, stating that the alternative
25
pathway acts by accommodating electrons that can not be handled by the
cytochrome pathway.
This
"energy overflow"
hypothesis has
been
extensively studied by Lambers and co-workers (Lambers, 1980) in respiration
of root tissue.
They found that the alternative pathway was only engaged
when there was an excess supply of carbohydrates to the roots relative to the
requirements for carbohydrate utilization. This behavior was also found in leaf
tissue (Azcon-Bieto et al., 1983).
In a similar way, seed respiration following a variety of stresses (aging,
chilling, solvents) has been shown to engage the cyanide-resistant pathway
when damage to the membranes selectively restricted the capacity of the
cytochrome pathway (Leopold and Musgrave, 1979). Low temperatures have
also been reported to stimulate the engagement of the alternative respiratory
capacity. In their studies of germinating corn seedlings Stewart et al. (1990a,
b) showed that alternative pathway capacity increased concomitantly with
respiration and was higher in mesocotyls grown at low temperatures. In their
view, low growth temperatures require the seedlings to have a higher
respiratory rate to prevent accumulation of toxic metabolites and the
alternative pathway functions in that respiration.
As stated early, the cyanide-resistant alternative pathway has been
considered a wasteful process, not involved in the production of energy
available to the plant (Lance et al., 1985). However some researchers have
suggested that this pathway is linked to some form of energy transduction
26
(Wilson, 1980).
cyanide
show
A common observation is that mitochondria resistant to
ADP/O
values
lower
than
those
of
cyanide-sensitive
mitochondria (Hackett et al., 1960; Lambowitz, 1972; Ducet, 1982).
Hackett et al. (1960) was the first to suggest that the low yield of ATP
in cyanide-resistant mitochondria was not due to a low efficiency of the
process
of
oxidative
phosphorylation
but
to
the
occurrence
of
nonphosphorylating electron transport pathway. A direct demonstration that
the alternative pathway is a nonphosphorylating pathway can be given by
using specific inhibitors to both pathways (SHAM and KCN). The addition of
cyanide, by curtailing the cytochrome pathway and diverting electrons to the
alternative pathway, causes a marked decrease in the efficiency of oxidative
phosphorylation.
No phosphorylation is associated with the oxidation of
succinate whereas one site remains associated with the oxidation of malate
(complex I site), because the alternative pathway branches at the ubiquinone
level (Lance et al., 1985).
In contrast to the evidence showing the
nonphosphorylating nature of the pathway, Wilson (1970, 1978, 1980) has
consistently reported some ATP production,
sensitive to
SHAM
and
oligomycin.
Residual respiration. Often even in the presence of both cyanide and
SHAM, Oj uptake is not always totally inhibited.
Assuming that the
concentrations used for cyanide and SHAM are efficient in completely
inhibiting the alternative and cytochrome oxidases, the presence of an active
27
oxidase other these oxidases has often been suggested (Amthor, 1989). This
component of O2 uptake has been called residual respiration. According to
Theologis and Laties (1978), this activity is not associated with the
mitochondria but involves extramitochondrial metabolism.
It therefore only
appears when using intact tissues, not isolated mitochondria.
Whether
residual respiration occurs in the absence of SHAM and cyanide is unknown
(Amthor, 1989).
Sesay et al. (1986) reported that in soybean residual
respiration can be as high as 10-20% and 42-44% for leaf discs of young and
mature leaves, respectively.
In roots of maize seedlings, Lambers and
Posthumus (1980) found residual respiration rates equivalent to 15% of
uninhibited respiration rate. In tissues of woody plants this phenomena is also
present. Cole et al. (1982) in studying the respiration of dormant pear flower
buds at 50C, indicated that up to 70% of oxygen flow was through the
alternative oxidase.
The presence of both SHAM and cyanide, inhibited
respiration only partially.
This residual respiration, whose nature was
unknown, was higher in P. calleryana than in P. communis.
Seed Respiration Studies
The respiratory metabolism of dormant seeds has been investigated in
several studies.
In the earliest ones, researchers only attempted to
characterize changes in the respiration rate of dormant seeds under
stratification (Pack, 1921; Brown, 1939; Barton, 1945; Vallance, 1952;
28
Nikolaeva, 1969).
However, the respiratory rate was determined not at
stratification temperatures but at room temperature or even higher (Nikolaeva,
1969). Other reports (Harrington, 1923; Visser, 1954; 1956a; 1956b) have
focused in the area of gas exchange in dormant seeds and its relationship to
temperature and pre-sowing treatment.
Pollock and Olney (1959) and Olney and Pollock (1960) reported
changes in respiratory activity as well as in levels of nitrogen and phosphorus
during stratification of sour cherry (Prunus cerasus) seeds. They found that
the capacity for growth developed linearly with time after 4 weeks of
stratification at 50C and paralleled an increase in dry weight in the embryonic
axis, indicating translocation of materials from the cotyledons, but was
preceded by cell divisions and a sharp rise in respiration rate. They proposed
that dormancy was imposed by a block in energy metabolism and was broken
by increased availability of phosphate acceptors such as glucose, via
hydrolysis of stored forms (Pollock and Olney, 1959).
According to them,
increased respiration was the key to the release from dormancy.
Many chemical inhibitors of respiration can also promote germination.
These inhibitors are known to block the terminal cytochrome oxidase of the
mitochondrial electron transport chain. As a consequence of this, Hendriks
and Taylorson (1972), proposed that the alternative respiratory (cyanideresistant) pathway was involved in the termination of dormancy. The action
of some other inhibitors of respiration that do not act directly on the electron
29
transport chain can likewise bring about germination in some species. This led
to the proposal that PPP was involved in some crucial step (Roberts, 1964a,
1964b, 1969).
The relative contribution of PPP and glycolysis pathways, to the
metabolic activity of stratifying sour cherry seeds was studied earlier by La
Croix and Jaswal (1967) using the C-6/C-1 ratio method. The initial high ratio
(0.95) was maintained during the first 6 weeks of stratification, then
decreased abruptly during the seventh week, and continued to decrease
gradually during the remainder of the stratification period.
They stated a
possible relationship between dormancy release and the activity of PPP.
There have been several reports relating PPP activity to the breaking of
dormancy, but the evidence for its involvement is still not clear. Bogatek and
Lewak (1988) investigated the activities of the PPP enzymes in apple seeds,
and found that termination of dormancy was connected with a change from
domination of PPP to glycolysis in sugar catabolism during cold stratification.
Cyanide pretreatment affected the C-6/C-1 ratio and the activities of the
enzymes under study in such a manner that the maxima of PPP and glycolysis
appeared early during stratification.
They suggested a regulatory role of
cyanide in the removal of dormancy (Bogatek and Lewak, 1988). The validity
of the C-6/C-1 ratio has been questioned because it does not account for
carbon diverted into storage compounds or sequestered as phytomass, and
for the same reason, the changes on it might not be as significant as they
30
appear to be (Bewley and Black, 1982).
Furthermore, Hu and Couvillon
(1990) questioned the role of PPP and catalase activity in dormancy control
of peach seeds as previously suggested by other researchers.
Their data
demonstrated that a significant increase in the activities of glucose-6phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase
(6PGDH), the two key enzymes in the PPP, occurred simultaneously with, but
not before, the increase in seed germination of stratified seeds. Germination
reached a maximum before peak activity levels of the two enzymes were
reached.
They concluded that the increase in PPP activity is probably the
result not the cause of dormancy breaking.
Bogateketal. (1991) found that HCN pretreatment not only stimulated
apple seed germination but prevented or alleviated some of the symptoms of
dormancy in non-cold treated embryos.
They then proposed a system in
which CN", from cyanogenic glucosides, plus Gibberellin A4, produced under
conditions favoring dormancy release, would act to remove embryonal
dormancy (Bogatek and Lewak, 1991).
Differential Scanning Calorimetry (DSC)
General Concepts
Measurement of heat evolution from plant tissues can constitute a
sensitive, non-invasive way to measure their rates of metabolic activity, and
estimate the relative efficiency of their systems at a particular stage of
31
development or under a determined set of environmental conditions (Loike et
al., 1981).
Nonetheless, until recently the measurement of heat evolution
was limited by available instrumentation.
The main principle behind all
calorimetric methods is the fact that whenever a material undergoes a change
in physical state, or whenever it reacts chemically, heat is either absorbed or
liberated (McNaughton and Mortimer, 1975).
this principle.
Living tissues do not escape
The heat evolved in these reactions can be accurately
quantified by the use of one or more differential thermal systems.
The
purpose of these is to record the difference between the enthalpy change
which occurs in a sample and that in some inert reference material, when they
are both kept at the same temperature. These systems can be classified into
three types: classical Differential Thermal Analysis (DTA), 'Boersma' DTA and
Differential Scanning Calorimetry (DSC).
According to McNaughton and
Mortimer (1975), the main difference between classical and 'Boersma' DTA
and DSC is that in the first two, both sample and reference are heated by a
single heat source. Temperatures are measured by sensors embedded in the
sample and reference material (classical), or attached to the pans which
contain the material (Boersma).
In DSC, sample and reference are each
provided with individual heaters, making possible the use of the "null-balance"
principle by two control loops.
One loop controls the sample and its
temperature according to a predetermined scanning rate, while the other
insures that no temperature differential is developed between the sample and
32
the reference by adjusting the power input to the reference (McNaughton and
Mortimer, 1975).
Modern DSC instruments work in two basic modes. In the isothermal
mode, the instrument is held at a given temperature, and the heat differential
between sample and reference represents a measurement of the heat of
metabolism of the former.
In the temperature-scanning mode, the
temperature of sample and reference changes at a programmed rate and the
heat difference between sample and reference changes at each temperature
(Hart Scientific, 1991).
Applications of DSC in Plant Respiration
Isothermal heat conduction calorimetry has been used to evaluate the
effect of temperature and oxygen depletion on metabolic rates of tomato and
carrot cell cultures by Criddle et al. (1988).
They reported that accurate
prediction of growth properties of intact plants was possible in these two
species, and that it represented a practical tool for germplasm screening and
selection. Criddle et al. (1990) studied the effect of NaCI on metabolic heat
evolution rates by barley roots. Two levels of inhibition by increasing salt
were found.
Following the transition from initial rate to the first level of
inhibition (50%), the decrease on metabolic activity was cultivar dependent
up to 150mM of salt.
At higher concentrations (>150mM) of salt,
metabolism was further decreased and no differences among the cultivars
33
were noted. Iversen et al. (1989) examined cut fresh pineapple metabolism,
and defined suitable storage conditions to avoid heat-producing spoilage
related with microbial growth.
Based on the metabolic heat rates of larch
shoot tips, Hansen et al. (1989) predicted long term growth of several clones
of this species.
Hansen and Criddle (1989) devised a batch injection attachment for
calorimeters, which permits environment modifications in the ampules without
affecting the DSC performance. Calorimetric studies do not allow the stirring
of cell cultures, and oxygen diffusion becomes a limiting factor. This problem
was solved by Fontana et al. (1990) by floating the cells on high density
media which allowed long time studies.
The effect of high and low
temperature stresses on the metabolic activity of plant tissues and cell
cultures were determined by Breidenbach et al. (1990). Their data show that
metabolic inactivation is a complex function of both temperature, and time of
exposure to the stress.
Simultaneous measurements of the rate of heat
generation, C02 production and Oj consumption were possible when Criddle
et al. (1990) devised a system compatible with commercial calorimeters.
In recent reports the use of differential scanning calorimetry to study
alternative respiration and heat evolution in plants has been proposed.
Ordentlich et al. (1991) reported the use of a DSC to measure heat evolution
from aging potato tubers and stressed cucumber leaves, two cases in which
cyanide resistant respiration has been studied by other methods. Skubats et
34
al. (1993) used DSC techniques to evaluate the effect of salicylic acid on heat
production in the spadix of the inflorescence of Sauratum guttatum and its
relation to alternative respiratory activity.
Different/a/ scanning calorimetry is a tool well suited for studying
sample responses over a temperature range, at a predetermined rate of
temperature change.
DSC studies of pea and soybean cotyledons allowed
Vertucci (1990) to study the thermodynamic properties of water in these
tissues.
Rank et al. (1991) measured decreasing metabolism of cultured
tomato cells as a result of exposures to high and low temperatures. Models
were used to describe thermal inactivation and prediction of activity loss,
following a stress. Gardea et al. (1993) studied metabolic activities during the
dormancy development of grape primary buds by means of isothermal as well
as temperature-scanning calorimetry.
They characterized the metabolic
efficiency of endo- and ecodormant buds in field and greenhouse conditions
using // (temperature coefficient of metabolism) as an indicator of the
metabolic stage of buds.
Analysis of Temperature-Scanning Data
The existence of an energy barrier requiring a minimum energy Umin to
cross is related to the concept of activation energy, which refers to the
minimum amount of energy necessary for some reaction to take place (Nobel
(1991).
At the end of the 19th century, Arrhenius proposed the following
35
equation to describe the rate of a chemical reaction (Montgomery and
Swenson, 1976; Nobel, 1991)
k=Ae-Ea"tT
where k is the rate constant, A is a constant, -Ea is activation energy, R is the
gas constant, and T is absolute temperature. A plot of the logarithm of the
rate constant versus 1/7" is commonly known as an Arrhenius plot.
Data
collected from scanning experiments can be analyzed this way. After several
assumptions were met, and appropriate substitutions made, Criddle et al.
(1992) modified the original equation revised by Johnson et al. (1974) to:
where q is the heat rate, C is a constant, and/y = E7R (using the nomenclature
of Johnson et al., 1974) is an apparent activation energy in Kelvin units.
Taking the natural log in both sides of the equation we obtain:
In^lnC-n/r
The slope of a line through data plotted as the natural log of the heat
rate versus the reciprocal of the Kelvin temperatures is equal to -//.
The
intercept is InC.
The justification for the use of// in complex biological reactions and its
interpretation in terms of catalytic efficiencies of enzymes is presented by
Johnson et al. (1974).
Temperature coefficients of biological reactions are
often reported as Q10 values (a unitless ratio of rates of reactions at one
36
temperature and at a temperature 10 degrees higher, often limited to a
particular range of temperature).
Q10 values are useful when comparing
effects of temperature but according to some authors have no "rational basis"
to allow interpretation on a mechanistic level (Johnson et al, 1974).
37
CHAPTER 3
METABOLIC ACTIVITIES OF PEACH SEEDS DURING DORMANCY
REMOVAL AND EARLY SEEDLING GROWTH
Abstract
The heat of metabolism (qmet) and CO2 evolution of 'Lovell' and
'Nemaguard' peach seeds stratified for 0 to
12 and 0 to 7 weeks,
respectively, and those of the seedlings grown from them were measured by
differential scanning calorimetry (DSC).
The temperature coefficient of
metabolism (//) of stratifying seeds was also measured.
increased with stratification time for both cultivars.
index of metabolic efficiency, was calculated.
qmet and C02
The ratio qmet/C02, an
A high ratio represents an
inefficiency of metabolism where a high proportion of the energy produced by
the tissue is lost as heat and not used for biomass production. For 'Lovell'
seeds, the index remained high and constant during the first two weeks,
decreased between 4 and 6 weeks and then remained constant. Therefore,
metabolic activity was low in unstratified and partially stratified seeds, but
increased after 6 weeks of stratification.
Eighty percent germination was
reached only after 6 weeks of stratification.
The time at 4C required for
germination was positively correlated with the qmet/C02 ratio whereas the
percentage of germination was negatively correlated.
Concurrent with
maximum germination at 6 weeks, metabolic efficiency was also at its
38
maximum. For 'Nemaguard', there was no correlation between the qmet/C02
ratio and germination percentages or number of days to germination.
The temperature coefficient of metabolism (//) decreased as temperature
increased but this change was dependant on cultivar and stratification
treatment. For 'Nemaguard', stratification treatment did not affect /v values
and these decreased as temperature increased regardless of the duration of
stratification.
For 'Lovell' seeds, /; values were highly dependant on
stratification time.
In both cultivars, increasing stratification time resulted in taller
seedlings, increased C02 evolution and, contrary to that observed in the
seeds, decreased qmet. For 'Lovell' seedlings the qmet/C02 ratio decreased with
stratification treatment and was inversely correlated with growth. For cv.
Nemaguard this correlation was not significant.
39
Introduction
Seeds of many woody plant species are usually dormant after fruit
ripening and will not germinate even if put under the proper environmental
conditions. These seeds need a period of stratification, that is incubation in
moist medium at temperatures ranging from 0 to 5C, from several weeks to
months in order to germinate and grow a normal phenotype (Nikolaeva,
1969). Excised embryos from non-stratified seeds have limited germinative
capacity and give rise to abnormal plants (physiological dwarfs). Such plants
have short internodes, epinastic leaf development and in many cases poor root
growth (Flemion, 1959; Tukey and Carlson, 1945; Wang and Beardow, 1968;
Frisby and Seeley, 1993a). The germinative capacity of the seeds and the
growth capacity of the resulting young seedlings increase with time in
stratification,
however the relationship between germination of seeds
(cessation of dormancy) and physiological dwarfing is not well understood.
The respiratory metabolism of dormant seeds has received the attention
of several studies. Stratification treatments have been shown to bring about
large increases in respiratory activity (Pollock and Olney, 1959; La Croix and
Jasval, 1967). Other studies have focused on the activities of glycolysis and
pentose phosphate pathways during stratification (La Croix and Jasval, 1967;
Bogatek and Lewak, 1988; Hu and Couvillon, 1990). No reports are available
relating respiratory changes during seed stratification with the metabolic
activity of the resultant seedlings and seedling vigor.
40
The measurement of heat evolution from plants tissues constitutes a
sensitive, non-invasive way to measure their rates of metabolic activity (Loike
et al, 1981). Also, the relative efficiency of plant metabolism at a particular
stage of development or under a given set of environmental conditions can be
estimated (Criddle et al., 1990).
Recent uses of differential temperature-
scanning and isothermal calorimetry have provided valuable information about
the effects of oxygen depletion on metabolic rates of cell cultures (Criddle et
al., 1988); high and low temperature stress on plant tissues and cell cultures
(Breidenbach, 1990); heat production and alternate respiratory activity in
thermogenic and non-thermogenic tissues (Ordentlich et al., 1991; Skubats
et al., 1993). Recently, Gardea et al. (1993) characterized metabolic changes
during the dormancy development of primary grape buds, using the
temperature coefficient of metabolism (//) and the ratio qmet/C02 evolution, an
index of respiratory efficiency, to study the pattern of respiratory changes
during this period and determine changes in the efficiency of plant
metabolism.
Studies trying to predict the long term growth of larch clones by
calorimetric measurements of metabolic heat rates were done by Hansen et
al. (1989). They proposed that heat of metabolism, measured under similar
conditions and stage of development, would give a general measure of
integrated metabolic rates that could predict growth rate potential of individual
trees. Their studies showed that even though there is a wide variation in heat
41
rates among larch clones, in some populations the metabolic rates could be
used to select trees with fast growth rate.
In this study, we report the use of isothermal and differential
temperature-scanning calorimetry to describe metabolic changes occurring
during seed stratification of two peach cultivars with different chilling
requirements and the seedlings arising from those treatments. By using this
approach, changes in the qmet, C02 evolution, the ratio qmet/C02 evolution and
JJ
were determined.
Our objective was to establish if the increase in
germinative capacity observed in peach seeds after stratification was related
to heat of metabolism and respiratory efficiency and to determine if these
changes if any persisted after germination of the seeds and were related to
physiological dwarfing.
42
Materials and Methods
Plant Materials
Seeds
of peach
(Prunus persica
(L.)
Batsch.,
cvs.
Lovell
and
Nemaguard) were used throughout these studies. The seeds, with endocarp
removed, were surface sterilized with a 75% v/v ethanol solution for 30 sec,
rinsed in distilled water, rinsed in a 1 % sodium hypochlorite solution for 15
min, and rinsed again with double distilled water (DDH20). For stratification,
the surface-sterilized seeds were placed on two layers of Whatman No. 3 filter
paper, moistened with DDH20 and placed in a 4C cold room in the dark for 0
to 12 weeks for 'Lovell' or 0 to 7 weeks for 'Nemaguard'.
Prior to
stratification, an incision was made along the cotyledonary gap. Groups of
seeds were prepared at different times, so that all stratification treatments
ended simultaneously. This provided a constant supply of seeds with different
degrees of chilling and a similar growth environment for germination and
seedling growth studies.
Evaluation of Germination and Seedling Growth
Stratified seeds were placed in 10 cm disposable petri dishes, filled with
moist filter paper. Three replications with 10 seeds to a dish were used for
each stratification treatment. The seeds were germinated at 23C in the dark.
A seed was considered germinated when radicle growth was 2 mm.
43
After radicle emergence, seeds were planted in cylindrical plugs (2.5 x
16 cm) in a medium of 1 peat: 1 vermiculite : 0.5 perlite, by volume. Seeds
from each petri dish were planted 2 cm deep in randomized rows of 10 seeds
each and moved to a greenhouse maintained at 25/20C (day/night). A 16 hr
photoperiod was provided by natural sunlight, supplemented with artificial
light by 1,000 W high pressure sodium lamps. The plugs were watered with
tap water as needed.
Four weeks after planting, primary stem height was measured from the
cotyledonary insertion to the terminal meristem.
Calorimetric Measurements
Metabolic-heat-rate measurements were made in a Hart model 4207
differential scanning calorimeter (Hart Scientific, Pleasant Grove, UT 84062).
The instrument has a baseline noise of ± 1 //Watt and a working range of -30
to 11OC. The temperature around the measuring chamber was maintained at
15C with a Fisher 9500 refrigerated circulating bath (Fisher Scientific,
Pittsburgh, PA 15230) using 30% v/v ethylene glycol as refrigerant. A flux
of argon was used to prevent moisture condensation in experiments below
room temperature.
Samples were measured in Hastelloy-C ampules with
removable lids and an approximate volume of 1 cm3.
Isothermal experiments measured heat rates at 24C for at least 3,000
sec; data were collected at 20 sec intervals. Heat of metabolism rates were
44
expressed on a fresh weight basis.
Measurements of CO2 evolution,
metabolic heat rate and calculations of metabolic efficiency were done
according to Criddle et al. (1990, 1991). These methods employ the insertion
of a C02-trapping solution, 0.4M NaOH, into the calorimeter ampule during
isothermal experiments to allow measurement of heat of reaction of C02 with
base and, therefore, C02-production rates. The NaOH was placed in a small
glass vessel to ensure the sample did not come in contact with the base. The
heat of reaction of CO2 with the NaOH solution was assumed to be constant
(-108.5 kJ-mol"1) at the temperature used in this study.
The efficiency of
metabolism was calculated as the metabolic heat rate divided by the rate of
COj production.
Temperature-scanning experiments measured metabolic rates over the
temperature range from 4 to 60C at a rate of S.BC-hr"1 with data acquisition
at 20 sec intervals (Hansen and Criddle, 1989). Rates of heat of metabolism
were expressed on a fresh weight basis. Arrhenius plots of the natural log of
heat rates vs. the reciprocal of the absolute temperature (xlOOO) were used
to determine // at 5C intervals from 5 to 40C (Fontana et al, 1990; Johnson
et al, 1974,; Montgomery and Swenson, 1976; Nobel, 1991).
Sample Preparation
Three replicates of two seed pieces (embryo + 0.8 cm of cotyledons)
or two 1 cm excised shoot apices were used for calorimetric measurements.
45
While preparing the equipment, seeds (after removal from the 4C chamber)
and excised apices were stored briefly in a petri dish on moist filter paper.
Statistical procedures
Changes in seed heat of metabolism, C02 evolution and the ratio
qmet/C02 evolution were analyzed as a completely randomized design using 3
replications per treatment.
Germination percentages were analyzed in the
same way but were calculated from 3 replicates of 10 seeds each. Seedling
height was calculated from a minimum of 15 seedlings per replicate, fj values
were calculated from a minimum of 3 replications.
46
Results and Discussion
Germination and Respiratory Metabolism
Germination of 'Lovell' seeds was about 10% for seeds stratified for 0
or 2 weeks, increased up to 20% by 4 weeks. By 6 weeks of stratification
germination percentage reached 80% and by 12 weeks was 100% (Fig.
3.1 A).
The number of days to germination decreased as stratification
proceeded, reaching minimal levels (2 days) only after 10 weeks. The qmet
values for unstratified 'Lovell' seeds was 0.62 //J/sec-mg FW, gradually
increased reaching 1.71 //J/sec-mg FW for fully stratified seeds.
Seeds
stratified for 0 to 4 weeks evolved similar C02 rates and increased to a
platform of 1.47 //mol/sec • mg FW x 10"6 after 6 weeks of stratification. COj
evolution was higher in the following stratification period from 6 to 10 weeks
and reached 6.26 //mol/sec-mg FW x 10"6 after 12 weeks of stratification.
The ratio qmet/C02 evolution was high and not significantly different during the
first 2 weeks, decreased until 8 weeks, and thereafter remained low and
constant.
Germination of 'Nemaguard' seeds was about 40% after 0,1, or 2
weeks of stratification (Fig. 3.2A), but after 3 weeks of stratification,
germination reached 80%. For unstratified seeds the average number of days
to germination was 11.5, and it decreased with longer stratification times to
reach a minimum of 4 days after 7 weeks of chilling.
47
The qmet value for unstratified and briefly stratified 'Nemaguard' seeds
was 0.17 //J/sec-mg FW and increased until 7 weeks to reach 0.95
//J/sec-mg FW. Levels of COj evolution were 0.59 and 0.63 //mol/sec-mg
FW x 10"6 for unstratified and 1-week stratified seeds, and reached 2.70
//mol/mg-sec FW x 10"6 after 7 weeks of stratification.
The ratio qmet/C02
evolution was highly variable for unstratified seeds remaining constant at
about 450 kJ/mol form 2 to 5 weeks of stratification and then declined.
The metabolic activity of dormant or partially stratified seeds was lower
than that of fully stratified seeds for both cultivars. This is consistent with
previously reported data reporting COj evolution or oxygen consumption rates
(Pollock and Olney, 1959; La Croix and Jasval, 1967; Bogatek and Lewak,
1988).
Dormant seeds evolved lower amounts of C02 and had lower heat
of metabolism than partially stratified seed (Figs. 3.IB and 3.2B).
The
metabolism of dormant and partially stratified seeds, based on qmet/C02 data,
is less efficient when compared to later stages of stratification.
A high qmet/C02 ratio represents an inefficiency of metabolism in which
energy produced by the plant is lost as heat and not used for biomass
production. The causes for the change from large to smaller values of this
ratio can be explained as a major change in metabolic pathways or an
inefficiency in energy coupling. In both cases, the amount of energy trapped
as ATP in relation to that portion lost as heat will change. As a reference, the
complete aerobic combustion of glucose is an exothermic process generating
48
approximately 455 ±15 kJ/mol. Similar values were obtained for seeds of cv.
Nemaguard, but much higher values for unstratified seeds of 'Lovell'.
Peach seeds have appreciable amounts of lipids as storage reserves.
In apple seeds, lipid reserves gradually decrease during the stratification
treatments, concomitant with an increase in starch formation (ZarskaMaciejewska and Lewak, 1976; Lewak and Rudnicki, 1977). The embryo of
these seeds contains both acidic and alkaline lipase activities, indicating that
the glyoxylate pathway could be an important source for gluconeogenesis at
this stage. The reactions of this pathway generate NADH and FADH2 that are
further oxidized by 02, there is a considerable amount of heat produced and
no C02 evolved. Thus, the ratio qmet/C02 evolution would be high. As the
lipid content on the seeds decrease, the activity of the glyoxylate cycle would
be lower, and glucose would become the main respiratory substrate (lowering
the qmet/C02 evolution ratio) and confirming the possibility of a change in the
metabolic pathways operating during stratification of 'Lovell' seeds.
The second possibility can be understood as an impaired respiratory
system in which there is a deficiency in electron transport acceptors (e.g
cytochrome c, as is the case in early-imbibed peanut seeds) (Wilson and
Bonner, 1971).
In this case, phosphorylation is only loosely coupled to
oxygen consumption and respiration is therefore inefficient and less than
maximal ATP production occurs.
In peach seeds, the energy block in the
metabolism would persist past early inhibition, probably indicating a bigger
49
impairment in the system that would be removed only as stratification
proceeds.
As metabolic efficiency increased (or qmet/C02 decreased), germination
percentage increased and time to germination decreased. The qmet/C02 ratio
of 'Lovell' seeds was significantly higher during the first 4 weeks of
stratification and was positively correlated with the number of days to
germination
and
negatively
correlated
with
germination
percentages.
However, the qmet/C02 ratio of 'Nemaguard' seeds was highly variable during
the first two weeks of stratification and then remained constant and was not
significantly correlated to germination percentage or number of days to
germination.
Temperature-scanning Experiments
Temperature effects on the rate of heat of metabolism of stratifying
seeds are presented in figures 3.3A and 3.4A In these figures heat evolution
rates, corrected by fresh weight, increased from 5C reaching maximum levels
at temperatures ranging from 39 to 44C for both cultivars.
Maximum
metabolic heat produced after each stratification treatment was significantly
different for both cultivars and reached maximum levels of 433.01 and
283.55 jj\N/mg FW after 12 and 7 weeks for 'Lovell' and 'Nemaguard' seeds,
respectively (Table 3.1).
There was no significant difference for the
temperature at which maximum heat was reached for 'Lovell' seeds (Break
50
temperature) (Table 3.1). For 'Nemaguard', longer stratification treatments
resulted in higher break temperatures.
The slopes of the curves of scanning data plotted in the Arrhenius form
represent apparent activation energies that constitute different metabolic
barriers for the metabolism to proceed (Fig 3.3B and 3.4.B). The temperature
coefficient of metabolism (//) for both cultivars was calculated from these
figures and is presented in figures 3.5A and 3.5B.
For 'Lovell' (Fig 3.5A)
there was a significant interaction between the range of measurement and the
stratification treatment (Appendix 1).
/J values
were lower as the temperature
range increased but the rate of decrease was different for each duration of
stratification. For the 5-1OC range , shorter stratification times resulted in
higher/y values, ranging from 31.4 to 10.0K for 0 and 12 weeks, respectively.
At higher temperatures differences between stratification treatments were less
pronounced (15.62 and 6.61 K for 0 and 12 weeks) and values were of the
same magnitude starting with the 15-20C range.
For 'Nemaguard', there
were no significant differences between the stratification treatments and no
interaction between stratification and temperature range was detected
(Appendix 2). The data presented in Fig. 3.5B corresponds to the mean //
values across stratification treatments.
The // value for the lowest
temperature range was 19.20, and decreased as temperature increased to
reach levels of 2.03K for fully chilled seeds (7 weeks).
51
The temperature-scanning studies show that the metabolism of
unstratified seeds is highly dependent on temperature at the lower
temperature ranges (Fig. 3.5). This dependance decreases with increasing
temperature but is related to cultivar and stratification time.
Nemaguard
seeds, independent of their stratification time, showed lower // values than
'Lovell' seeds and gradually decreased across the ranges of temperature
included in the study. These data are consistent with the qmet/C02 values and
would indicate that for this cultivar only one metabolic pathway is active
during the dormancy removal though stratification. For the cv. Lovell, at the
lower temperature range (5-10), unstratified seeds showed the highest /;
values and fully stratified seeds the lowest, indicating a possible change in the
metabolic pathways active at those temperatures for those seeds that have
had longer stratification treatments.
As in the case of 'Nemaguard' this
temperature dependence of the metabolism decreases with increasing
temperatures and differences among stratification times disappear. Young
(1990) calculating activation energies (Ea) from COj evolution of dormant
apple buds also found that Ea decreases with increasing temperatures and
chilling accumulation. Ea changes were explained as a shift in the source of
the COj evolved or a possible low temperature membrane alteration that either
induced or predisposed changes in the enzymes involved in the respiratory
process (Young, 1990; Wang and Faust, 1987).
52
Seedling Height and Respiratory Metabolism
Seedling height at 4 weeks after planting (Figs. 3.6A and 3.7A)
increased with longer stratification times for both cultivars. The number of
seedlings showing leaf lesions and rosette-type growth decreased as
stratification time for the respective seeds increased, and it was significantly
lower for 'Nemaguard' seedlings (data not shown).
Due to the low
germination percentage of seeds stratified for 0 to 1 week, there were no
seedlings available for this part of the experiment or for the measurement of
seedling metabolic activity.
Calorimetric measurements of seedling shoot apices for 4-weeks old
seedlings are presented in figures 3.6B and 3.7B.
qmet values for 'Lovell'
ranged between 5.33 and 5.66 //J/sec-mg FW in seedlings coming from
seeds stratified for 2 to 6 weeks.
By 8 weeks, qmet decreased to 4.13
/yj/sec-mg FW and remained constant in the remaining stratification
treatments. C02 evolution remained at the same level for the first 8 weeks,
ranging from 6.96 to 7.15 ;vmol/sec • mg FW x 10"6. Seedlings apices excised
from seeds stratified for 10 and
12 weeks evolved 8.95 and 9.67
A/mol/sec-mg FW x 10"6, respectively. The ratio qmet/C02 evolution was high
for those seedlings developing from seeds receiving 2 to 6 weeks of
stratification and decreased to reach its lowest levels for those seedlings
derived from 12 weeks of stratification.
53
qmet values for 'Nemaguard' were of similar magnitude as those for
'Lovell' seedlings, but did not have as evident a decrease in qmet as the
duration
of stratification
increased.
C02 evolution was
about 6.50
/ymol/sec • mg FW x 10"6 for seedlings derived from seeds with 2 to 4 weeks
of stratification.
constant.
These values increased until 6 weeks and then remained
The ratio qmet/C02 was high for the first two weeks, gradually
decreased to 570.5 kJ/mol, and remained constant for the rest of the
stratification.
The efficiency of the metabolic activity of peach seedlings was affected
by the length of seed stratification.
Seedlings from shorter stratification
treatments showed the highest qmet/C02 values, indicating an inefficiency of
the metabolism, as did the seeds from which they were grown. The ratio was
inversely correlated to seedling height of 'Lovell' seedlings. Consistent with
the seed data, the ratio for seedlings of the cv. Nemaguard significantly
changed only after 6 weeks of stratification.
There was no correlation
between seedling growth and the ratio qmet/C02.
The removal of the effects of physiological dwarfing of seedlings has
been attempted using several methods.
Gibberellin acid applications or
exposure to a long photoperiod (Flemion, 1959) have only temporarily
improved
growth of such seedlings.
Only the exposure to
chilling
temperatures (between 5 and 7C) has been effective in obtaining normal
growth
and development of dwarfed seedlings (Davidson, 1 934; Flemion,
54
1959; Frisby and Seeley, 1993b). For these reasons it is not surprising that
the metabolic impairment detected in peach seeds persists in the seedlings.
Differences between cultivars relative to germination and seedling
growth have been attributed to a differential sensitivity to inhibitors and
promoters of germination (Mehanna et al., 1985) rather than to the direct
action of these growth regulators. We cannot speculate about that in this
report but the changes in metabolic efficiency reported here perhaps would
help illustrate the idea that the block in germination and growth is a block in
energy availability and that treatments that remove it do so by altering the
respiratory metabolism of the cell.
The studies reported here provide evidence for chilling having an effect
on the respiratory metabolism of peach seeds during stratification treatment
and indicate a change that is not only reflected in the metabolic properties of
the stratifying seeds but also in the resulting seedlings. The identification of
the metabolic pathways active during the removal of seed dormancy and early
seedling growth should provide insight on the mechanism involved in
controlling germination and early seedling growth of seeds. The importance
of using at least two cultivars with different chilling requirements in this type
of study is noted.
55
Table 3.1.
Maximum heat evolution and break temperatures from
temperature scanning experiments with peach seeds of cultivars
Lovell and Nemaguard, stratified at 4C for 0 to 12 and 0 to 7
weeks respectively2.
Cultivar
Parameter
Lovell
Nemaguard
z
Stratification
time (weeks)
Maximum heat
rate (/yW/mg FW)
Break temperature
(C)
0
102.85 a
41.18
2
156.73 ab
41.26
4
201.25 be
43.09
6
257.80 c
42.60
8
367.20 d
43.55
10
370.72 de
42.97
12
433.01 e
40.81
***
NS
0
89.23 a
40.63 cb
1
119.01 ab
39.68 ab
2
122.79 ab
39.08 a
3
148.16 ab
40.89 cb
4
175.85 b
41.88 c
5
256.50 c
44.16 d
6
283.55 c
43.57 d
7
280.74 c
43.60 d
**»
*
Lower case letters indicate significant differences at P = 0.05 according to
Waller-Duncan test.
NS,
'"'Nonsignificant, or significant at P = 0.05 and 0.001 respectively.
56
1000
o
E
CM
o
o
e
E
J200
4
6
8
10
Weeks at 4C
Figure 3.1.
Days to germination and percentage of germination (A) and (B)
changes in heat of metabolism (qmet), CO2 evolution rates and in
the qmet/C02 evolution ratio of 'Lovell' peach seeds stratified at
4C for 0 to 12 weeks.
57
18
—1
C
O
16
■A
4=
<0
14
~
.£
12 -
i©
O*
1
1
1
I
T
/ r
1
90
-
/
/
>■
4
c
40
E
10
0
I
20
1
1
-
.
t
1
I
1
*
1
0
^
B
8
1.2
■
0)
CO
-5 0.8
3
^
u
\./
10.4
o-
1 v
1
I
1
0
1
\
s
■ 6
»>£^ ■ 4
is^^^- 2
/
■^
0.0
#
CD
1
1000
►
- 10 o~ .
800
D)
E
o
(0
50
y
o:
^.20
"
I
? 1.6
C
30 |
2
o
<£
60 %
4
,
BJ
80
- 70
_
Q
100
^-*
$—^
'
^
1/^ i
>
1N
o
8
o
1
E
C O) .
.2 E
5 6
O JO
600 3
CM
■ 400
o
O
Wo
M
E
(j Ct
200
0
"
^^
0
2
*"-
=5
3
4
5
6
o
V
7
Weeks at 4C
Figure 3.2.
Days to germination and percentage of germination (A) and (B)
changes in heat of metabolism (qmet), CO2 evolution rates and in
the qmet/C02 evolution ratio of 'Nemaguard' peach seeds stratified
at 4C for 0 to 7 weeks.
58
0(0). 2 (•), 4 (V). 6 (▼). 8(0). 10(B). 12(A)
Weeks
500
400
(0
03
»
x:
JO
aa
300
Am
o E
.Q
»,
A
200
(0
<D
.o
S
«««vvv_
^.
100
Jgg>
10
20
30
40
50
A.
60
3.5
3.6
Temperature (C)
05
0)
s:
o
"o
(0
V
4-»
E
3.0
3.1
3.2
3.3
3.4
1000/T(K)
Figure 3.3.
Differential scanning calorimetric determination of the metabolic
heat rates of 'Lovell' peach seeds stratified for 0, 2, 4, 6, 8, 10,
and 12 weeks at 4C. Direct calorimetric data (A) and Arrhenius
plots (B).
59
0(0), i (•). 2(v). 3(T), 4(D), 5(m). 6(A), 7(A) Weeks
500
400
(0
^
o
U-
300
o O)
n E 200
(0
*->
*
*
£
A
**.■■••■■••$
*
•
-oooooan
=s.
100
»*.'Io'>5*i;»",V«ooooooooooo ""•r%:
■■lifi"
10
20
30
40
50
60
Temperature (C)
3.2
3.3
3.4
1000/T (K)
Figure 3.4.
Differential scanning calorimetric determination of the metabolic
heat rates of 'Nemaguard' peach seeds stratified for 0, 1, 2, 3,
4, 5, 6, and 7 weeks at 4C. Direct calorimetric data (A) and
Arrhenius plots (B).
60
0(0), 2 (•), 4<V),
6(T),
8(D),
10(H),
12(A)
Weeks
35
30
^
25
£ 20
W
"5 15
CD
E
C 35
<D
O 30
B
H-
0)
O 25
O
>I 20
2 15
Q.
E 10
•-
5
in
o
CM
I
If)
1
o
m
m
CM
o
CM
o
m
m
o
to
o
1
CM
Scanning range (C)
Figure 3.5.
Temperature coefficients of metabolism (//) of peach seeds cv.
Lovell stratified for 0, 2, 4, 6, 8, 10, and 12 weeks at 4C (A) and
average temperature coefficient of metabolism for
'Nemaguard' seeds stratified for 0 to 7 weeks (B).
61
T
1000
800
- 600
O
E
O
U
a
400
E
200
4
6
8
10
12
Weeks at 4C
Figure 3.6.
Seedling height (A) and (B) changes in heat of metabolism (q^),
C02 evolution rates and in the qmet/C02 evolution ratio for
seedlings obtained from seeds of cv. Lovell stratified at 4C for
2 to 12 weeks.
62
-i 1000
800
600
400
o
E
C4
o
o
E
J 200
Weeks at 4C
Figure 3.7.
Seedling height (A) and (B) changes in heat of metabolism (qmet),
COa evolution rates and in the qmet/C02 evolution ratio for
seedlings obtained from seeds of cv. Nemaguard stratified at 4C
for 2 to 7 weeks.
63
Literature Cited
Bogatek, R. and S. Lewak. 1988. Effects of cyanide and cold treatment on
sugar catabolism in apple seeds during dormancy removal. Physiol.
Plant. 73:406-411.
Breidenbach, R.W., D.R. Rank, A.J. Fontana, L.D. Hansen, and R.S. Criddle.
1990. Calorimetric determination of tissue responses to thermal
extremes as a function of time and temperature. Thermochim. Acta
172:179-186.
Criddle, R.S., R.W. Breidenbach, D.R. Rank, M.S. Hopkin, and L.D. Hansen.
1990. Simultaneous calorimetric and respirometric measurements on
plant tissues. Thermochim. Acta. 172:213-221.
Criddle, R.S., A.J. Fontana, D.R. Rank, D. Paige, L.D. Hansen, and R.W.
Breidenbach. 1991. Simultaneous measurements of metabolic heat rate,
COj production, and 02 consumption by microcalorimetry. Annals
Biochem. 194:413-417.
Criddle, R.S., R.W. Breidenbach, E.A. Lewis, D.J. Eatough, and L.D. Hansen.
1988. Effects of temperature and oxygen depletion on metabolic rates
of tomato and carrot cell cultures and cuttings measured by calorimetry.
Plant, Cell and Environment 11:695-701.
Davidson, O.W. 1934. Growing trees from 'non-viable' peach seeds. Proc.
Amer. Soc. Hort. Sci. 32:308-312.
Flemion, F. 1959. Effect of temperature, light and gibberellic acid on stem
elongation and leaf development in physiologically dwarfed seedlings
of peach and rhodotypos. Contrib. Boyce Thompson Inst. 20:57-70.
Fontana, A.J., L.D. Hansen, R.W. Breidenbach, and R.S. Criddle. 1990.
Microcalorimetric measurements of aerobic cell metabolism in unstirred
cell cultures. Thermochim. Acta 172:105-113.
Frisby, J.W. and S. Seeley. 1993a. Chilling of endodormant peach propagules.
II. Initial seedling growth. J. Amer. Soc. Hort. Sci. 118:253-257.
Frisby, J.W. and S. Seeley. 1993b. Chilling of endodormant peach propagules.
III. Budbreak and subsequent growth of physiologically dwarfed to near
normal seedlings. J. Amer. Soc. Hort. Sci. 118:258-262.
64
Gardea, A.A., Y.M. Moreno, A.N. Azarenko, P.B. Lombard, and L.S. Daley.
1993. Changes in metabolic properties of grape buds during
development. J. Amer. Soc. Hort. Sci. (Accepted)
Hansen, L.D., E.A. Lewis, D.J. Eatough, D.P. Fowler, and R.S. Criddle. 1989.
Prediction of long term growth rates of larch clones by calorimetric
measurements of metabolic heat rates. Can. J. For. Res. 19:606-611.
n, L.D., and R.S. Criddle. 1989. Batch-injection attachment for the Hart
Hansen,
differential scanning calorimeter. Thermochim. Acta 154:81-88.
Hu, H. and G.A. Couvillon. 1990. Activities of catalase and pentose
phosphate pathway dehydrogenases during dormancy release in
nectarine seed. J. Amer. Soc. Hort. Sci. 115:987-990.
Johnson, F.H., H. Eyring, and B. Jones. 1974. The theory of rate processes
in biology and medicine, pp. 550. John Wiley and Sons. New York.
La Croix, J.L. and A.S. Jaswal. 1967. Metabolic changes in after-ripening
seed of Prunus cerasus. Plant Physiol. 42:479-480.
Lewak, S. and R.M. Rudnicki. 1977. After-ripening in cold-requiring seeds. In:
The physiology and biochemistry of seed dormancy and germination.
A.A. Khan, ed. Elsevier/North-Holland, Amsterdam, pp. 193-217.
Loike, J.D., S.C. Silverstein, and J.M. Sturtevant. 1981. Application of
differential scanning microcalorimetry to the study of cellular processes:
Heat production and glucose oxidation of murine macrophages. Proc.
Natl. Acad. Sci. U.S.A. 78(10):5958-5962.
Mehanna, H.T., G.C. Martin, and C. Nishijima. 1985. Effects of temperature,
chemical treatments and endogenous hormone content on peach seeds
germination and subsequent seedling growth. Scientia Hort. 27:63-73.
Montgomery, R. and C.A. Swenson. 1976. Quantitative problems in the
biochemical sciences (2nd edition) pp. 370. W.H. Freeman and Co.New
York.
Nikolaeva, M.G.I 969. Physiology of deep dormancy in seeds. Israeli program
for scientific translations. Jerusalem, pp. 146-158.
Nobel, P.S. 1991. Physicochemical and Environmental Plant Physiology.
Academic Press. New York. 635 pp.
65
Ordentlich, A.; R.A. Linzer and I. Raskin. 1991. Alternative respiration and
heat production in plants. Plant Physiol. 97:1545-1550.
Pollock, B.M. and H.O. Olney. 1959. Studies of the rest period. I. Growth,
translocation and respiratory changes in the embryonic organs of the
after-ripening cherry seed. Plant Physiol. 34:131-142.
Skubats, H. and B.J. Meeuse. 1993. Energy loss in tissue slices of the
inflorescence of Sauromatum guttatum (Schott) analyzed by
microcalorimetry. J. Exp. Bot. 44:493-499.
Tukey, H.B. and R.F. Carlson. 1945. Morphological changes in peach
seedlings following after-ripening treatments of the seeds. Bot. Gaz.
106:431-440.
Wang, D. and J. Beardow. 1968. Thermo-, photo-effect on rosaceous seeds
during germination as expressed in subsequent seedling development.
Contrib. Boyce Thompson Inst. 24:17-23.
Wang, S.Y. and M. Faust. 1987. Metabolic activities during dormancy and
blooming of deciduous fruit trees. Israel J. Bot. 37:227-243.
Wilson, S.B. and W.D. Bonner. 1971. Studies of electron transport in dry and
imbibed peanut embryos. Plant Physiol. 48:340-344.
Young, E. 1990. Changes in respiration rate and energy of activation after
chilling and forcing dormant apple trees. J. Amer. soc. Hort. Sci.
115(5):809-814.
Zarska-Maciejewska, B. and S. Lewak. 1976. The role of lipases in the
removal of dormancy in apple seeds. Planta 132:177-181.
66
CHAPTER 4
CHANGES IN THE CAPACITIES OF THE CYTOCHROME AND
ALTERNATIVE RESPIRATORY PATHWAYS MEASURED BY DIFFERENTIAL
SCANNING CALORIMETRY DURING THE STRATIFICATION OF PEACH
SEEDS
Abstract
The capacities of cytochrome and alternative respiratory pathways in
the embryo of peach (Prunuspersica L. Batsch. cv. Loveil) were investigated
by differential scanning calorimetry. Heat evolution measurements showed
a progressive increase in the total respiratory capacity (measured at 24C) of
isolated embryos as the stratification period increase. Respiratory inhibitor
studies showed a significant respiratory capacity through the alternative
pathway of respiration and present during the entire stratification treatment.
Cytochrome pathway activity increases steadily during stratification, becoming
significantly higher than alternative pathway capacity after 6 weeks. The rise
in cytochrome pathway capacity is significantly correlated to the increase
germination percentage and seedling height. Alternative pathway capacity
was not correlated to seedling height but it was significantly correlated to
germination percentage.
67
Introduction
Dormant seeds of many woody plant species need a period
incubation
in
moist medium at temperatures ranging from 0 to
of
5C
(stratification), from several weeks to months in order to germinate and grow
a normal phenotype (Nikolaeva, 1969).
Many metabolic and morphological
changes occurring during stratification have been characterized for several
seed species (Lewak at al.,
Thevenot, 1982).
during
1975;
Dawidowicz-Grzegorzewska,
1980,
Among these, the respiratory metabolism of the seeds
stratification
has
received
the
attention
of
several
studies.
Stratification has been shown to bring about large increases in respiratory
activity (Pollock and Olney, 1959; La Croix and Jasval, 1967). However, the
intensity
of
apple seed
respiration
measured
at the temperature
of
stratification is relatively stable increasing only at the end of the stratification
(Bogatek and Lewak, 1978). Tissaoui and Come (1973) found that dormancy
breaking and germination of apple seeds can occur under anaerobic conditions
and questioned that increased respiratory activity is necessary for dormancy
removal.
Most of the studies concerning the respiratory metabolism of dormant
seeds have focused on the activities of glycolysis and pentose phosphate
pathways during stratification (La Croix and Jasval, 1967; Bogatek and
Lewak, 1988; Hu and Couvillon, 1990). Alscher-Herman et al., (1981) and
Bogatek and Rychter (1984) investigated the respiratory activity of pear and
68
apple seeds during stratification and attempted to determine the respiratory
pathways (e.g. cytochrome and alternative) active during this period. They
found a progressive increase in respiratory capacity during seed stratification
but concluded that this increase was due to the cytochrome pathway and that
the alternative pathway of respiration was not involved on it.
However the alternative (cyanide-resistant) respiratory pathway may
play a role in the respiratory metabolism during dormancy termination and
germination of seeds (Yentur and Leopold, 1976; De la Fuente-Burguillo and
Nicolas, 1977; James and Spencer, 1979; Esashi et al., 1981, 1982; Adkins
et al. 1984; Stewart et al., 1990a). Seeds of many plant species, including
Prunus spp. contain cyanogenic glucosides that upon the action of Bglucosidase liberate hydrogen cyanide.
The HCN liberated may inhibit the
operation of the cytochrome pathway of respiration and, as a result, increase
the flux of electrons through the CN-resistant pathway.
Current methods for evaluating alternative respiratory activity are based
on oxygen uptake measurements in the presence and absence of specific
respiratory inhibitors of the pathway (e.g. salicylhydroxamic acid, SHAM)
(Schonbaum, et al., 1971). A more sophisticated approach is based on the
discrimination of stable oxygen isotopes by the alternative and cytochrome
pathways (Guy et al., 1989).
The measurement of heat evolution from plant tissues constitutes a
sensitive, non-invasive way to measure their rates of metabolic activity, and
69
estimate the relative efficiency of their systems at a particular stage of
development or under a given set of environmental conditions (Loike et al.,
1981; Criddle et al., 1990). Differential temperature-scanning and isothermal
calorimetry have provided valuable information on effects of oxygen depletion
on metabolic rates of cell cultures (Criddle et al., 1988); prediction of long
term growth of seedlings (Hansen et al., 1989); high and low temperature
stress on plant tissues and cell cultures (Breidenbach, 1990); characterization
of dormancy development of buds (Gardea et al., 1993) and changes in
metabolic activities of peach seeds during dormancy removal and early
seedling growth (Chapter 3).
Two recent reports have used calorimetric
measurements for the study of alternative respiratory activity (Ordentlich et
al., 1991; Skubats et al., 1993).
In these studies heat of metabolism and
alternative respiratory activity were measured in thermogenic and nonthermogenic tissues.
In this study we report the use of differential scanning calorimetry to
describe changes in respiratory activity and determine the capacities of the
cytochrome and alternative respiratory pathways during stratification of peach
seeds, and their possible relationship to seedling growth.
70
Materials and Methods
Plant Materials
Seeds of peach {Prunus persica (L.) Batsch., cv. Lovell) were surface
sterilized with a 75% v/v ethanol solution for 30 sec, rinsed in distilled water,
rinsed in a 1 % sodium hypochlorite solution for 15 min, and rinsed again with
double distilled water (DDHjO). For stratification, the surface-sterilized seeds
were placed on two layers of Whatman No. 3 filter paper, moistened with
DDHjO and placed in a 4C cold room in the dark for 0 to 12.
Prior to
stratification, an incision was made along the cotyledonary gap. Groups of
seeds were prepared at different times and then placed in the 4C chamber, so
that all stratification treatments ended simultaneously.
This provided a
constant supply of seeds with different degrees of chilling and a similar
growth environment for germination and seedling growth studies.
Sample Preparation
Three replicates of five embryos each (approx. 40 mg of tissues) were
used for calorimetric measurements.
Seeds were taken out of the 4C
chamber and while setting up the experiment, were stored briefly in petri
dishes with moist filter paper. Embryos were removed and incubated under
vacuum with inhibitor solutions for 2 hr before placing them in the calorimeter
ampules.
To determine the effects of sodium azide (NaNg) on respiratory
71
activity, embryos were incubated in 0, 105, 10"4, 10"3, 102M sodium azide in
the absence/presence of 10"3M SHAM. Reverse activity response data was
obtained with incubation in 0,
10'4,
10"3, and 10"2M SHAM in the
presence/absence of 10"3M sodium azide. Preliminary experiments were done
to determine most appropriate length of the incubation period and to ensure
that
neither
incubation
nor
vacuum
infiltration
interfered
with
the
determination of the activities of the cytochrome or alternative pathways.
Calorimetric Measurements
Metabolic-heat-rate measurements were made in a Hart model 4207
differential scanning calorimeter (Hart Scientific, Pleasant Grove, UT 84062).
The instrument has a baseline noise of ± 1 ^/Watt and a working range of -30
to 110C. The temperature around the measuring chamber was maintained at
15C with a Fisher 9500 refrigerated circulating bath (Fisher Scientific,
Pittsburgh, PA 15230) using 30% v/v ethylene glycol as refrigerant. A flux
of argon was used to prevent moisture condensation in experiments below
room temperature.
Samples were measured in Hastelloy-C ampules with
removable lids and an approximate volume of 1 cm3.
Isothermal experiments measured heat rates at 24C for at least 3,000
sec; data were collected at 20 sec intervals.
expressed on a fresh weight basis.
Metabolic heat rates were
72
Respiratory Activity Data and Statistical Procedures
Heat of metabolism data were obtained for 0, 2, 4, 6, 8, 10, and 12
weeks of stratification and analyzed as a completely randomized design with
3 replications per stratification treatment.
Respiratory pathway capacities
were determined as described by Vanlerberghe and Mclntosh (1992).
Cytochrome pathway (CP) capacity was taken to be that portion of metabolic
heat evolution inhibited by 10"3M of azide in the presence of 10"3I\/I SHAM,
and alternative pathway (AP) capacity was taken to be that portion of
metabolic heat evolution inhibited by 10"3M SHAM in the presence of 10'3M
azide.
73
Results
To estimate the extent to which the cytochrome oxidase and alternative
respiratory pathways are operative in stratifying seeds, measurements of the
influence of SHAM (an alternative pathway inhibitor) (Schonbaum et al.,
1971) and of sodium azide (a cytochrome oxidase inhibitor) on heat evolution
in peach seed embryos were made. The effect of different concentrations of
sodium azide on heat evolution of peach embryos during stratification is
presented as a surface response in Fig. 4.1.
At 0 weeks of stratification
maximum inhibition of heat evolution (ca. 14% of initial level) was only
obtained with the maximum concentration of azide used in the study (Fig
4.1 A). Lower concentrations of azide did not inhibit heat evolution and on the
contrary increased it by as much as 50% compared to the uninhibited
controls.
After 2 weeks of stratification, uninhibited heat evolution was
similar to that of the unstratified controls.
At this time there was no
stimulation of metabolic activity by sodium azide and metabolic activity was
only partially inhibited (ca. 20%). A similar level of inhibition was obtained in
embryos stratified for 4 weeks.
From
that point on, uninhibited heat
evolution rates increased up to 12 weeks of stratification.
In embryos
stratified for more than 4 weeks, sodium azide inhibited respiratory activity by
about 40-50%.
Upon addition of 10"3M SHAM, inhibition by sodium azide increased for
all stratification treatments (Fig.4.IB).
Note that in this case the control
74
treatment corresponds to the heat rate obtained with no sodium azide but in
the presence of 10"3M SHAM as compared to Fig 4.1 .A, in which the control
represents heat evolution in the absence of SHAM or sodium azide. Residual
respiratory activity, measured as that part of the metabolic heat evolution
present after incubation with sodium azide + SHAM, was low and variable,
ranging from ca. 17% for unstratified seeds to 7% for seeds stratified for 10
weeks.
The effect of SHAM alone and of SHAM + sodium azide on heat
evolution is presented in Fig. 4.2. SHAM alone (Fig 4.2A) had only a small
effect on heat of metabolism, and it was most inhibitory on unstratified
embryos with 35% inhibition. For the remaining treatments, inhibition was
on average 15% at the 10"3M concentration. Higher SHAM concentrations
severely inhibited heat evolution.
These results are not surprising since
concentrations higher that 2mM SHAM have been reported to have significant
effects on other processes of the cell (Moller et al., 1988).
Upon addition of 10"3M sodium azide, heat evolution was inhibited by
as much as 84% of the control (containing 103M SHAM) for unstratified
embryos (Fig 4.2B). The percent of inhibition decreased as stratification time
increased. Residual heat evolution was very high at 12 weeks of stratification
(up to 43.7% of the uninhibited heat evolution rates).
The capacities of both respiratory pathways are presented in Fig 4.3.
This figure shows an steady increase in both cytochrome and alternative
75
pathway capacity with stratification time. However during the first 4 weeks
of stratification the capacity for the alternative pathway is higher than that of
the cytochrome pathway. After that cytochrome pathway capacity increases
and is higher than that of the alternative pathway for the remaining
stratification treatments.
The activities of both pathways were positively correlated to the mean
percentage of germination and negatively correlated to the number of days to
germination (Table 4.1).
Cytochrome pathway activity was significantly
correlated to seedling height (P=0.0161), whereas the activity of the
alternative pathway was not {P = 0.0576).
76
Discussion
Seeds that exhibit endodormancy are in a state in which germination is
prevented. The mechanism by which this temporary cessation of growth is
overcome is not well known. In many cases if seeds germinate without total
dormancy release, the resultant seedlings will grow morphologically abnormal
(Flemion, 1934; Come, 1970).
Stratification treatments are effective in dormancy removal, and as
reported in this experiments, produce a steady and substantial rise in the
metabolic activity of peach embryos. Based on the results obtained using the
respiratory inhibitors presented here, this increase in respiratory capacity
includes a measurable rise in the capacity for respiration through the
alternative pathway, but the main component after 7 weeks of stratification
appears to be the cytochrome oxidase pathway of respiration.
Alscher-
Herman et al. (1981) reported a similar rise in total respiratory capacity of
stratifying pear seeds with increasing stratification, but they concluded that
it did not include a rise in alternative respiration.
The estimation of the degree of participation of the alternative pathway
in the metabolic activity of tissues using respiratory inhibitors has some
limitations (Alscher-Herman et al., 1981). The specificity of SHAM for the
alternative oxidase has been questioned by Rich et al. (1978) who found that
hydroxamates inhibit a selection of well known enzymes (e.g. tyrosine,
peroxidase, lipoxygenase) with very low KjS.
In fact. Parish and Leopold
77
(1978) warned about mistaking lipoxygenase activity for alternative pathway
activity.
Most of the concerns about the use of SHAM for determining
alternative pathway activity arise from the use of oxygen uptake
measurements as indicators of respiration.
When measuring respiratory
activity as heat evolution using calorimetry, additional oxygen uptake by other
oxidases should not interfere with the results.
As reported earlier (Chapter 3), calorimetric determinations of metabolic
efficiency of stratifying peach seeds show low values of efficiency during the
first 4 weeks of cold stratification, with values increasing markedly only after
6 weeks of stratification. This change in efficiency, measured as a decrease
in the amount of metabolic heat divided by COj evolution, suggest that a
change in metabolic pathways occurs during stratification.
This is in
agreement with the higher amount of heat evolved if the alternative
respiratory pathway were active during the early stages of stratification.
Perhaps more important than the alternative respiratory capacity during
the first weeks of stratification (that may be responsible for an important part
of the metabolic activity at this time) the results of our experiments show that
the increase in capacity of the cytochrome c oxidase mediated pathway is
coincident with the increased germination percentages and the decrease in
seedling abnormalities observed in seeds stratified for 6 weeks (Fig. 3.1.A,
Table 4.1). If the seedlings obtained from partially stratified seeds have an
impaired cytochrome oxidase pathway that may explain the dwarfism they
78
exhibit. In maize, genetic dwarfs arise from a mutation in one of the units of
the cytochrome c oxidase complex. In the case of peach embryos, the genes
could be present but not expressed at this time or the enzyme assembly
mechanism may be impaired.
A rise in cytochrome c oxidase activity (the chief enzyme in the
cytochrome pathway), has been found to occur upon imbibition of some
species that do not undergo a dormancy period before germinating (Nawa and
Asahi, 1971; Matsuoka and Asahi, 1983). The proposed mechanism for the
resumption of rapid respiratory activity does not include de novo synthesis of
the subunits of the enzyme, but the assemblage of these pre-formed units to
form the active enzyme.
De novo nuclear and mitochondrial gene
transcription, as well as mRNA translation is initiated only about the first 12
hr of embryo germination (Matsuoka and Asahi, 1983; Ehrenshaft and Brambl,
1990).
A similar mechanism may mediate the increase in cytochrome pathway
respiratory capacity observed during seed stratification in peach. However,
the low respiratory efficiency of the first weeks of stratification and the
significant alternative respiratory capacity during this period suggest a role for
cyanide-resistant respiration during the early stratification in peach. The net
energy output (ATP production) of the alternative pathway is thought to be
negligible although some reports show evidence to the contrary (Wilson,
1970, 1978, 1980). If the alternative respiratory pathway does not supply
79
energy to a peach embryo during stratification, perhaps its role, as others
have proposed, is to serve as an overflow mechanism for the cytochrome
oxidase pathway which shows limited activity during the initial stratification
period.
80
Table 4.1.
Correlations of the mean cytochrome and alternative pathway
activities and the number of days to germination, germination
percentage and seedling height for peach seeds cv. Lovell,
stratified for 0 to 12 weeks.
Days to
germination
Germination
percentage
Seedling
height
Cytochrome pathway
activity
-0.9809
(P = 0.0001)
0.99014
(P=0.0001)
0.89461
(P = 0.0161)
Alternative pathway
activity
-0.93725
(P=0.0018)
0.9139
{P =0.004)
0.83719
[P = 0.0576)
81
10
Figure 4.1.
-2
Effect of sodium azide (NaNg) concentrations in the absence (A)
or presence (B) of 10'3M SHAM on the rate of heat evolution at
24C of peach seeds stratified at 4C for 0 to 12 weeks. Each line
intersection of the grid represents a mean of three replicates.
82
^v
Figure 4.2.
Effect of salicylhydroxamic acid (SHAM) concentrations in the
absence (A) or presence (B) of 10'3M sodium azide on the rate of
heat evolution at 24C of peach seeds stratified at 4C for 0 to 12
weeks. Each line intersection of the grid represents a mean of
three replicates.
83
Cytochrome pathway (• )
2
4
Alternative pathway (V)
6
8
10
Weeks at 4C
Figure 4.3.
Cytochrome and alternative pathway respiratory capacity of
peach seeds stratified at 4C for 0 to 12 weeks. Each
point represents a mean of three replicates ± SE
84
Literature Cited
Adkins, S.W., G.M. Simpson and J.M. Naylor. 1984. The physiological basis
of seed dormancy in Avena fatua. IV. Alternative respiration and
nitrogenous compounds. Physiol. Plant. 60:234-238.
Alscher-Herman, R., M. Musgrave, A.C. Leopold, and A.A. Khan. 1981.
Respiratory changes with stratification of pear seeds. Physiol. Plant.
52:156-160.
Bogatek, R. and S. Lewak. 1978. Respiratory processes during apple seed
after-ripening. Acta Physiol. Plant. 1:45-51
Bogatek, R. and A. Rychter. 1984. Respiratory activity of apple seeds during
dormancy removal and germination. Physiol. Veg. 22:181-191.
Bogatek, R. and S. Lewak. 1988. Effects of cyanide and cold treatment on
sugar catabolism in apple seeds during dormancy removal. Physiol.
Plant. 73:406-411.
Breidenbach, R.W., D.R. Rank, A.J. Fontana, L.D. Hansen, and R.S. Griddle.
1990. Calorimetric determination of tissue responses to thermal
extremes as a function of time and temperature. Thermochim. Acta
172:179-186.
Come, D. 1970. Les obstacles a la germination. Masson et cie. Paris, pp.5480.
Criddle, R.S., R.W. Breidenbach, D.R. Rank, M.S. Hopkin, and L.D. Hansen.
1990. Simultaneous calorimetric and respirometric measurements on
plant tissues. Thermochim. Acta. 172:213-221.
Criddle, R.S., R.W. Breidenbach, E.A. Lewis, D.J. Eatough, and L.D. Hansen.
1988. Effects of temperature and oxygen depletion on metabolic rates
of tomato and carrot cell cultures and cuttings measured by calorimetry.
Plant, Cell and Environment 11:695-701.
Dawidowicz-Grzegorzewska, A. 1980. Anatomy, histochemistry and cytology
of dormant and stratified apple embryos. Ill Structural changes during
early development of seedlings in relation to embryonal dormancy. New
Phytol. 87:573-579.
85
De la Fuente-Burguillo, P., and G. Nicolas. 1977. Appearance of an alternate
pathway cyanide-resistant during germination of seeds of Cicer
arietinum. Plant Physiol. 60:524-527.
Ehrenshaft, M. and R. Brambl. 1990.
Respiration and mitochondrial
biogenesis in germinating embryos of maize. Plant Physiol. 93:295-304.
Esashi, Y., H. Komatsu, R. Ushizawa, and Y. Sakai. 1982. Breaking of
secondary dormancy in cocklebur seeds by cyanide and azide in
combination with CjHU and 02 and their effects on cytochrome and
alternative respiratory pathways. Aust. J. Plant Physiol. 9:97-111.
Esashi, Y., Y. Sakai and R. Ushizawa. 1981. Cyanide-sensitive and cyanideresistant respiration in the germination of cocklebur seeds. Plant
Physiol. 67:503-508.
Flemion, F. 1934. Dwarf seedlings from non-after-ripening embryos of peach,
apple and hawthorn. Contrib. Boyce Thompson Inst. 6:205-209.
Gardea, A.A., Y.M. Moreno, A.N. Azarenko, P.B. Lombard, and L.S. Daley.
1993. Changes in metabolic properties of grape buds during
development. J. Amer. Soc. Hort. Sci. (Accepted)
Guy, R.D., J.A. Berry, M.L. Fogel, and T.C. Hoering. 1989. Differential
fractionation of oxygen isotopes by cyanide-resistant and cyanidesensitive respiration in plants. Planta. 177:483-491.
Hansen, L.D., E.A. Lewis, D.J. Eatough, D.P. Fowler, and R.S. Criddle. 1989.
Prediction of long term growth rates of larch clones by calorimetric
measurements of metabolic heat rates. Can. J. For. Res. 19:606-611.
Hu, H. and G.A. Couvillon. 1990. Activities of catalase and pentose
phosphate pathway dehydrogenases during dormancy release in
nectarine seed. J. Amer. Soc. Hort. Sci. 115:987-990.
James, T.W. and M.S. Spencer. 1979. Cyanide-insensitive respiration in pea
cotyledons. Plant Physiol 64:431-434.
La Croix, J.L. and A.S. Jaswal. 1967. Metabolic changes in after-ripening
seed of Prunus cerasus. Plant Physiol. 42:479-480.
Lewak, S., A. Rychter, and B. Zarska-Maciejewska. 1975. Metabolic aspects
of embryonal dormancy in apple seeds. Physiol. Veg. 13:13-22.
86
Loike, J.D., S.C. Silverstein, and J.M. Sturtevant. 1981. Application of
differential scanning microcalorimetry to the study of cellular processes:
Heat production and glucose oxidation of murine macrophages. Proc.
Natl. Acad. Sci. U.S.A. 78(10):5958-5962.
Matsuoka, M. and T. Asahi. 1983. Mechanism of the increase in cytochrome
c oxidase activity in pea cotyledons during seed hydration. Eur. J.
Biochem. 134:223-229.
Moller, M., A. Berczi, L.H. van der Plas and H. Lambers. 1988. Measurement
of the activity and capacity of the alternative pathway in intact plant
tissues: Identification of problems and possible solutions. Physiol. Plant.
72:642-649.
Nawa, Y. and T. Asahi. 1971. Rapid development of mitochondria in pea
cotyledons during the early stage of germination. Plant Physiol.
48:671-674.
Nikolaeva, M.G. 1969. Physiology of deep dormancy in seeds. Israeli program
for scientific translations. Jerusalem, pp. 146-158.
Ordentlich, A.; R.A. Linzer and I. Raskin. 1991. Alternative respiration and
heat production in plants. Plant Physiol. 97:1545-1550.
Parish, D.J. and A.C. Leopold. 1978. Confounding of alternate respiration by
lipoxygenase activity. Plant Physiol. 62:470-472.
Pollock, B.M. and H.O. Olney. 1959. Studies of the rest period. I. Growth,
translocation and respiratory changes in the embryonic organs of the
after-ripening cherry seed. Plant Physiol. 34:131-142.
Rich, P.R., N.K. Wiegand, H. Blum, A.L. Moore, and W.D. Bonner. 1978.
Studies on the mechanism of inhibition of redox enzymes by substituted
hydroxamic acids. Biochim. Biophys. Acta. 525:325-337.
Schonbaum, G.R., W.D. Bonner, Jr., B.T. Storey, and J.T. Bahr. 1971.
Specific inhibition of the cyanide-insensitive respiratory pathway in
plant mitochondria by hydroxamic acids. Plant Physiol. 47:124-128.
Skubatz, H. and B. Meeuse. 1993. Energy loss in tissue slices of the
inflorescence of Sauromatum guttatum (Schott) analyzed by
microcalorimetry. J. Exp. Bot. 44:493-499.
87
Stewart, C.R.; B.A. Martin; L. Reding and S. Cerwick. 1990a. Respiration and
alternative oxidase in corn seedling tissues during germination at
different temperatures. Plant Physiol. 92:755-760.
Thevenot, C. 1982. Correlations between axis and cotyledons in apple seeds
embryo dormancy. Z. Pflanzenphysiol. 106:13-26.
Tissaoui, T. and D. Come. 1973. Levee de dormance de I'embryon de
pommier (Pyrus ma/us L.) en ('absence d'oxygene et de froid. Planta
111:315-322.
Vanlerberghe, G.C. and L. Mclntosh. 1992. Coordinate regulation of
cytochrome and alternative pathway respiration in tobacco. Plant.
Physiol. 100:1846-1851.
Wilson, S.B. 1970. Energy conservation associated with cyanide-insensitive
respiration in plant mitochondria. Biochim. Biophys, Acta.
223:383-387.
Wilson, S.B. 1978. Cyanide-insensitive oxidation of ascorbate + NNN'N'tetramethyl-p-phenylene-diamine mixture by mung bean (Phaseolus
aureus) mitochondria. Biochem. J. 176:129-136.
Wilson, S.B. 1980. Energy conservation by the plant mitochondrial cyanideinsensitive oxidase. Some additional evidence. Biochem. J.
190:349-360.
Yentur, S., and A.C. Leopold. 1976. Respiratory transition during seed
germination. Plant Physiol. 57:274-276.
88
CHAPTER 5
METABOLIC EFFICIENCY OF STRATIFYING PEACH SEEDS IS AFFECTED
BY GIBBERELLIN TREATMENTS
Abstract
The effects of GA3 application after differential stratification of peach
seeds on the respiratory metabolism was investigated by differential scanning
calorimetry. Heat of metabolism (qmet) measurements confirmed the increase
in respiratory capacity with stratification time with untreated controls. GA3
treatment increased qmet and COj evolution rates after 2 and 4 weeks of
stratification when compared to untreated controls.
The ratio qmet/C02
evolution, an index of respiratory efficiency was low for unstratified seeds,
indicating a very efficient metabolism; increased after 2 weeks of stratification
and returned to low levels only after 7 weeks. GA3 treatment prevented the
loss of respiratory efficiency observed early in the stratification period. Values
of qmet/C02 evolution were comparable to those of fully stratified control
seeds.
Even though GA3 enhanced both respiratory efficiency and
germination, its effects on seedling growth did not completely replaced the
chilling requirement for the obtention of a normal phenotype.
89
Introduction
Mature seeds of most peach cultivars achieve full germination capacity
only after 6 to 12 weeks of cold stratification. Full stratification completely
removes all hindrances preventing the germination and normal growth of
seedlings (Come, 1970).
Removing all or a portion of the seed coat, or
puncturing it, permits germination of peach seeds without chilling (Tukey,
1933; Flemion, 1934; Lammerts, 1942; Mehanna and Martin, 1985; Chao
and Walker, 1966). However the resulting seedlings are stunted, indicating
that some barriers to reach full growth potential still exist.
Another treatment to remove dormancy is by application of growth
regulators, that would supposedly change the hormonal equilibrium of the
seed (Lewak, 1981). However, such treatments do not completely eliminate
the symptoms of dormancy in some species (Bradbeer, 1968; Lewak, 1981;
Pinfield and Davies, 1978). Gibberellins eliminate the chilling requirement for
peach and apple seeds (Zigas and Coombe, 1977b; Rouskas et al., 1980).
Treatment of non-stratified seeds of two peach rootstocks with GA3
eliminated dwarfism symptoms in seedlings, but the results were dependant
on the chilling requirement of the genotypes (Mehanna et al., 1985).
GA3
treatment only increased seedling height of the cuitivar with a low chilling
requirement leaving the longer chilling requirement cuitivar stunted (Mehanna
et al., 1985).
90
Most of the work on the respiratory metabolism of stratifying seeds has
focused on the activities of the pentose phosphate or glycolytic pathways
(Roberts, 1969; Simmonds and Simpson, 1971; Hendricks and Taylorson,
1975; Kovacs and Simpson, 1976; Goslin and Ross, 1980; Bogatek and
Lewak, 1988) or the activity of the alternative respiratory pathway (AlscherHerman et al., 1981; Bogatek and Rychter, 1984). GA3 treatment increased
respiratory activity of germinating tomato seeds but the results may not be
totally relevant to peach seed physiology since tomato seeds have an
additional light requirement for germination that is not present in peach (Gui
et al., 1991).
The simultaneous measurement of metabolic heat (qmet) and C02
evolution rates constitutes a sensitive, non-invasive way to measure rates of
metabolic activity and estimate the relative efficiency of respiration at a
particular stage of development or under a given set of environmental
conditions (Loike et al., 1981; Criddle et al., 1990, 1991).
Recent uses of
differential temperature-scanning and isothermal calorimetry for the study of
plant metabolism have been discussed elsewhere (Chapter 2).
In this study we report the effects of GA3 treatment after varying
stratification times on the heat of metabolism, COj evolution and respiratory
efficiency of peach (Prunus persica L. Batsch, cv. Lovell) seeds and its
relationship to germination and seedling growth.
91
Materials and Methods
Plant Materials
Seeds of peach (Prunus persica (L.) Batsch, cv. Lovell) were used
throughout these studies. The seeds, with endocarp removed, were surface
sterilized with a 75% v/v ethanol solution for 30 sec, rinsed in distilled water,
rinsed in a 1 % sodium hypochlorite solution for 15 min, and rinsed again with
double distilled water (DDH20). For stratification, the surface-sterilized seeds
were placed on two layers of Whatman N03 filter paper, moistened with
DDH20 and placed in a 4C cold room in the dark for 0 to 12 weeks. Prior to
stratification, an incision was made along the cotyledonary gap. Groups of
seeds were prepared at different times and then placed in the 4C chamber, so
that all stratification treatments ended simultaneously.
This provided a
constant supply of seeds with different degrees of chilling and a similar
growth environment for germination and seedling growth studies.
After
stratification, random samples of 10 seeds each were placed in disposable
petri dishes lined with filter paper and moistened with 5 ml of water or 500
mg I"1 GA3 for 0 to 5 days. This concentration was selected, based on
previous results by Mehanna et al. (1985) that suggested that this was the
most effective in stimulating peach seeds germination and seedling growth.
92
Evaluation of Germination and Seedling Growth
A minimum of two replications of 10 seeds each of GAg-treated and
controls were used for each stratification treatment. Seeds were germinated
at a constant 23C under dark conditions. A seed was considered germinated
when radicle growth was 2 mm. After radicle emergence, seeds were planted
in cylindrical plugs (2.5 x 16 cm) that contained soilless medium (1 peat : 1
vermiculite : 0.5 perlite, by volume). Seeds from each petri dish were planted
2 cm deep in randomized rows of 10 seeds each.
Greenhouse conditions
were maintained at 25/20C (day/night). A 16 hr photoperiod was provided
by natural sunlight, supplemented with artificial light by 1,000 W high
pressure sodium lamps. The plugs were watered with tap water as needed.
Four weeks after planting, primary stem height was measured from the
cotyledonary insertion to the terminal meristem.
Calorimetric Measurements
Three replicates composed of five embryos each (approx. 40 mg of
tissues) or 3 pieces of cotyledons (approx. 80 mg of tissue) were used for
calorimetric measurements. With the testa removed, embryos were separated
from cotyledons and while setting up the experiment, both were stored briefly
in petri dishes filled with moist filter paper. Metabolic heat rate measurements
were made in a Hart model 4207 differential scanning calorimeter (Hart
Scientific, Pleasant Grove, UT 84062). The instrument has a baseline noise
93
of ± 1 //W and a working range of -30 to 110C. The temperature around the
measuring chamber was maintained at 15C with a Fisher 9500 refrigerated
circulating bath (Fisher Scientific, Pittsburgh, PA 15230) using 30% v/v
ethylene glycol as the refrigerant.
A flux of argon was used to prevent
moisture condensation in experiments below room temperature.
Samples
were measured in Hastelloy-C ampules with removable lids and an
approximate volume of 1 cm3.
Isothermal experiments measured heat rates at 24C for at least 3,000
sec; data were collected at 20 sec intervals.
Metabolic heat rates were
expressed on a fresh weight basis. Measurements of metabolic heat (qmet) and
CO2 evolution rates, and calculations of metabolic efficiency were done
according to Criddle et al. (1990, 1991). C02 evolution was quantified by
insertion of a CC^-trapping solution, 0.4M NaOH, into the calorimeter ampule
during isothermal experiments to allow measurement of heat of reaction of
C02 with base and, therefore, CCVproduction rates. The NaOH was placed
in a small glass vessel to ensure the sample did not come in contact with the
base. The heat of reaction of COj with the NaOH solution was assumed to
be constant (-108.5 kJ • mol1) at the temperature used in this study. An index
of efficiency of metabolism was calculated as the metabolic heat rate divided
by the rate of C02 production. A high qmet/C02 ratio represents an inefficiency
of metabolism where the energy produced by the plant is lost as heat and not
used for biomass production.
94
Statistical Procedures
Changes in seed heat of metabolism, C02 evolution and the ratio
qmet/C02 evolution were analyzed as a completely randomized design using a
minimum of three replications per treatment. Germination percentages were
obtained as the number of seeds germinated divided by the initial number of
seeds, using between 12 and 20, depending on the material available.
95
Results
Germination of "Lovell" seeds stratified from 0 to 2 weeks was about
15%, increased steadily to 95% by 12 weeks (Fig 5.1 A).
GA3 treatment
enhanced germination of non-stratified or partially stratified seeds, and for all
stratification treatments, germination was greater than 80%.
GA3 treatment also stimulated seedling growth of non-chilled and
partially chilled seeds (Fig. 5.1 B). Seedling height was highest in those plants
grown from seeds subjected to longer stratification treatments.
A small
number of non-chilled, untreated seeds grew no more than 1 cm and died.
Seedling height was about 4 cm for those seedlings grown from seeds
stratified for 2 weeks, by 4 weeks growth potential had increased by 50%,
and by 7 weeks it had doubled. Maximum seedling height was observed after
12 weeks of stratification. GAg-treated unstratified seeds produced seedlings
that had a height equivalent to 7 weeks of stratification.
With longer
stratification time, seedling height increased and was higher than that of
untreated controls. After 12 weeks of stratification, GA3-treatment did not
increase seedling growth.
The qmet value for cotyledons of unstratified seeds was about 0.5
/yj/sec-mg FW, increased by 70% after 2 weeks of stratification and
remained constant until 7 weeks (Fig. 5.2A). By 12 weeks, qmet values were
3 times higher than those for unstratified controls. There was no significant
difference in the qmet values between controls and GAg-treated seeds.
In
96
terms of C02 evolution, untreated and GAg-treated cotyledons were not
significantly different during the entire stratification treatment with the
exception of 7 weeks of stratification (Fig. 5.2B).
Values for unstratified
seeds were about 2 //mol C02/sec • mg FW, remained constant during 4 weeks
of stratification, increased after 7 weeks and were 5 times higher after 12
weeks of stratification. The qmet/C02 evolution ratio was higher for cotyledons
of unstratified and partially stratified seeds and decreased after 7 weeks of
stratification (Fig 5.2C). The qmet/C02 ratio for GAg-treated cotyledons was
significantly lower than that of untreated controls during the first four weeks
of stratification, but at 7 weeks these differences disappeared.
The qmet value for embryos of unstratified seeds was about 1.3
fjj/sec-mg FW, doubled after 2 weeks of stratification and continued
increasing to reach 3.2 /yJ/sec-mg FW at 12 weeks of stratification (Fig.
5.3A).
qmet values for GAg-treated embryos were higher than those of the
untreated controls after 2 and 4 weeks of stratification and the same at 7 and
12 weeks.
A similar patter for C02 evolution was observed for embryos
where values for GAg-treated embryos after 2 or 4 weeks of stratification
were higher than those of untreated controls, and reached similar values after
7 or 12 weeks (Fig. 5.3B). The qmet/C02 ratio was significantly lower for GAgtreated embryos after 2 or 4 weeks of stratification. Unstratified seeds and
those stratified for 7 or 12 weeks and treated with or without GAg were not
different (Fig. 5.3C).
97
Discussion
Chilling plays an important role in overcoming dormancy of peach
seeds, providing a stimulus that permits increased germination and normal
seedling growth (Mehanna et al., 1985). GA3 treatment of non-chilled seeds
constitutes a way to compensate for lack of chilling, and as shown in these
experiments, to stimulate growth of the resultant seedlings. The increase in
germination after GA3 application has been reported earlier (Hundal and
Khajuria, 1979; Mehanna et al., 1985) but the effect of GA in stimulating
seedling growth has been dependant on the cultivar and the length of chilling
requirement of the genotype (Mehanna et al., 1985). Our results show that
even though GA3-treated unstratified seeds give rise to smaller seedlings than
fully stratified seeds, GA3 treatment has a significant effect in alleviating the
symptoms of physiological dwarfing in seedlings.
The increase in germination percentage and seedling growth is
coincident with the higher respiratory efficiency observed in the GAa-treated
embryos.
Although unstratified embryos respired at very low rates as
evidenced by the qmet and C02 evolution rates, they are highly efficient (Fig.
5.3C). As stratification progresses, respiratory efficiency (as reflected in the
qmet/C02 ratio) decreases for the following 4 weeks, only to improve again
after 7 weeks of stratification. This metabolic change can be explained as an
inefficiency in energy coupling or a major change in metabolic pathways,
where a higher proportion of the energy produced by respiration is lost as
98
heat. These data agree with our previous report (Gardea et al., 1993) that
showed a similar fall and rise pattern of respiratory efficiency during early
developmental stages of ecodormant grape buds. The respiratory efficiency
of grape buds just beginning to swell decreases, to increase again only after
buds are fully swollen. The role of GA3 treatment in the increase in percent
germination and seedling growth can be understood as increasing respiratory
efficiency in partially stratified seeds to the levels of fully stratified ones. The
efficiency values observed here are similar to those reported for the complete
oxidation of glucose to H20 and COj and would indicate that the metabolic
activity at this time is dominated mainly by the glycolytic pathway.
In general, the stimulation of germination and seedling growth through
GA3 treatments has been explained as counteracting the effect of endogenous
inhibitors (e.g. ABA) but the evidence for this is not conclusive {Mehanna et
al., 1985; Ross, 1985). Whether this putative relationship is associated with
metabolic efficiency is unknown for there are only a limited number of reports
concerning the control of respiratory activity by growth regulators (Amthor,
1989). GA treatments have been shown to stimulate growth of pea seedlings
by increasing the net inflow of sugars consumed by respiration to the epicotyl
(Miyamoto and Kamisaka, 1990). Others have proposed a role for exogenous
GA as removing dormancy of apple seeds by stimulating lipase activity that
would then hydrolyze storage lipids, initiating the transformation of fats into
99
sugars that subsequently increase the availability of substrates for glycolysis
(Zarska-Maciejewska et al., 1980; Bogatek and Lewak, 1988).
Endogenous CN could also play a role by stimulating the oxidative
pentose phosphate pathway (PPP) through inhibition of catalase activity
(Hendricks and Taylorson, 1975). Our own experiments in peach seeds of
this cultivar (Chapter 4) show a significant capacity for alternative respiration
during the early phase of stratification, that perhaps can provide an overflow
mechanism for the cytochrome pathway at this time (Lambers, 1980). Gui
et al. (1991) reported an increase in SHAM-resistant alternative respiratory
activity after GA treatment of tomato seeds previously incubated in
hydrochloric acid, reaching values of up to 64% of total respiratory activity.
In our opinion GA3 did not affect peach embryos similarly because significant
alternative respiratory activity would increase the qmet/C02 evolution ratio,
because of the higher amount of heat evolved relative to C02 production that
would occur in such case.
We can not rule out the possibility that GA3 treatments may directly or
indirectly affect other factors that may regulate seed germination and
subsequent seedling growth. For example the interaction of G3 with other
hormones, availability of respiratory substrates or respiratory pathways.
100
100
90
80
70
60
50
40
30
20
10
0
^9
C
o
■ •■■■
CO
*c
^^
1 ^
!=
0)
CD
4-*
x:
O)
"<D
^
^«^
= £
:= O
■u ^
O
0
CO
18
16
14
12
10
8
6
4
i^
2
0
J
L
0
2
6
8
J
1_
10
12
Weeks at 4C
Figure 5.1.
Germination percentages (A) and seedling height (B) for peach
seeds cv. Lovell treated with 0 or 500 mg • I"1 GA3, after
stratification at 4C for 0 to 12 weeks.
101
1
£
T
1
1
A
3.5
LL
r
1
r
• - GA
3.0
0+ GA
2.5
O)
sE
2.0
0)
V)
1.5
—i
3
1.0
E
•
0.5
0.0
14
<o
25
x"C O)
.2
E
+■>
D O
O 0)
> ^
ai 0
^E
Oo
^ E
?-)
fc
_v
B
12
10
8
6
4
2
0
800
700
600
500
400
300
200
100
0
0
2
4
6
8
10
12
Weeks at 4C
Figure 5.2.
Changes in heat of metabolism (qmet) (A), C02 evolution rates (B)
and in the qmet/C02 evolution ratio (C) of 'Lovell' peach
cotyledons treated with 0 or 500 mg-l'1 GA3, after
stratification at 4C for 0 to 12 weeks.
102
3.5
3.0
2.5
2.0
• - GA
OH- GA
1.5
CO
1.0
0.5
0.0
14
(O
o
^
X LI.
r O)
o
4-1
b
♦
^ o
o (1)
CO
>
LU o
o F5
o
CM
CM.—.
O o
tJ E
H
1
1
■i
H
-1
I
L_
8
10
12
h
B
12
10
8
6
4
2
0
800
700
600
500
400
300
200
100
0
'HH
■
■
i_
0
2
4
6
Weeks at 4C
Figure 5.3.
Changes in heat of metabolism (qmet) (A), C02 evolution rates (B)
and in the q^/COz evolution ratio (C) of 'Lovell' peach embryos
treated with 0 or 500 mg • I"1 GAg, after stratification at 4C for 0
to 12 weeks.
103
Literature Cited
Alscher-Herman, R., M. Musgrave, A.C. Leopold, and A.A. Khan. 1981.
Respiratory changes with stratification of pear seeds. Physiol. Plant.
52:156-160.
Amthor, J.S. 1989. Respiration and crop productivity. 218 pp. SpringerVerlag, New York.
Bogatek, R. and S. Lewak. 1988. Effects of cyanide and cold treatment on
sugar catabolism in apple seeds during dormancy removal. Physiol.
Plant. 73:406-411.
Bogatek, R. and A. Rychter. 1984. Respiratory activity of apple seeds during
dormancy removal and germination. Physiol. Veg. 22:181-191.
Bradbeer, J.W. 1968. The role of endogenous inhibitors and gibberellins in
dormancy and germination of Corylus avellana L. seeds. Planta.
78:266-278.
Chao, L. and D.R. Walker. 1966. Effect of temperature, chemicals and seed
coat on apricot and peach seed germination and growth. Proc. Am.
Soc. Hort. Sci. 88:232-238.
Come, D. 1970. Les obstacles a la germination. Masson et cie. Paris, pp.
54-80.
Criddle, R.S., R.W. Breidenbach, D.R. Rank, M.S. Hopkin, and L.D. Hansen.
1990. Simultaneous calorimetric and respirometric measurements on
plant tissues. Thermochim. Acta. 172:213-221.
Criddle, R.S., A.J. Fontana, D.R. Rank, D. Paige, L.D. Hansen, and R.W.
Breidenbach. 1991. Simultaneous measurements of metabolic heat rate,
COj production, and 02 consumption by microcalorimetry. Annals
Biochem. 194:413-417.
Flemion, F. 1934. Dwarf seedlings from non-after-ripening embryos of peach,
apple and hawthorn. Contrib. Boyce Thompson Inst. 6:205-209.
Gardea, A.A., Y.M. Moreno, A.N. Azarenko, P.B. Lombard, and L.S. Daley.
1993. Changes in metabolic properties of grape buds during
development. J. Amer. Soc. Hort. Sci. (Accepted)
104
Goslin, P.G. and J.D. Ross. 1980. Pentose phosphate metabolism during
dormancy breakage of Corylus avellana L. Planta 148:362-366.
Gui, M.X., X.M. Wang, and W.Y. Huang. 1991. The effect of light and
exogenous gibberellic acid on respiration pathways during germination
of tomato seeds. Physiol. Plant. 81:403-407.
Hendricks, S.B. and R.B. Taylorson. 1975. Breaking of seeds dormancy by
catalase inhibition. Proc. Natl. Acad. Sci. USA. 72:306-309.
Hundal, P.S. and H.N. Khajuria. 1979. Effect of GA3 and thiourea on seed
germination of different varieties of peach. Indian J. Agric. Sci.
49:417-419.
Kovacs, M.I.P., and G.M. Simpson. 1976. Dormancy and enzyme levels in
seeds of wild oats. Phytochemistry 15:455-458.
Lambers, H. 1980. The biological significance of cyanide-resistant respiration
in higher plants. Plant Cell Env. 3:293-302.
Lammerts, W.E. 1942. Embryo culture an effective technique for shortening
the breeding cycle of deciduous trees and increasing germination of
hybrid seeds. Am. J. Bot. 29:166-171.
Lewak, S. 1981. Regulatory pathways in removal of apple seed dormancy.
Acta Hort. 120:149-159.
Loike, J.D., S.C. Silverstein, and J.M. Sturtevant. 1981. Application of
differential scanning microcalorimetry to the study of cellular processes:
Heat production and glucose oxidation of murine macrophages. Proc.
Natl. Acad. Sci. U.S.A. 78(10):5958-5962.
Mehanna, H.T. and G.C. Martin. 1985. Effect of seed coat on peach seed
germination. Scientia Horticulturae 25:247-254.
Mehanna, H.T., G.C. Martin, and C. Nishijima. 1985. Effects of temperature,
chemical treatments and endogenous hormone content on peach seed
germination and subsequent seedling growth. Scientia Horticulturae.
27:63-73.
Miyamoto, K. and S. Kamisaka. 1990. Effect of gibberellic acid on epicotyl
growth and carbohydrate distribution in derooted Pisum sativum
cuttings with or without cotyledons. Physiologia Plantarum.
80:357-364.
105
Pienfield, N. J. and H.V. Davies. 1978. Hormonal changes during after-ripening
of Acer platanoides L. seeds. Z. Pflanzenphysiol. 90:171-191.
Roberts, E.H. 1969. Seed dormancy and oxidation processes. In: Dormancy
and survival. Symp. Soc. Exp. Biol. 23:161-192.
Ross, J.D. 1985. Metabolic aspects of dormancy. In: Seed physiology. Vol 2.
Germination and reserve mobilization. D.R. Murray (Ed.), pp 45-75.
Academic Press. Orlando, FL.
Rouskas, D., J. Hugard, R. Jonard, and P. Villemur. 1980. Contribution a
I'etude de la germination des graines de Pecher (Prunuspersica Batsch.)
cultivar INRAGf305: effets de la benzyl-amino-purine (BAP) et les
gibberellines GA3 et GA4+7 sur la levee de dormance embryonnaire et
('absence des anomalies foliaires observees sur les plantes issues de
graines non stratifiees. C.R. Acad. Sci. Paris, 291:861-864.
Simmonds, J.A. and G.M. Simpson. 1971. Increased participation of PPP in
response to after-ripening and gibberellic acid treatment in caryopses
of Avena fatua. Can. J. Bot. 49:1833-1840.
Tukey, H.B. 1933. Effect of photoperiod and temperature on growth of
embryo cultured seedlings. Am. J. Bot. 30:707-711.
Zarska-Maciejewska, B.; I. Sinska; E. Witkowska, and S. Lewak. 1980. Low
temperature, gibberellin and acid lipase activity in removal of apple
dormancy. Physiol. Plant. 48:532-535.
Zigas, R.P. and B.G. Coombe. 1977b. Seedling development in peach, Prunus
persica (L.) Batsch. II. Effect of plant growth regulators and their
possible role. Aust. J. Plant. Physiol. 4:359-369.
106
BIBLIOGRAPHY
Adkins, S.W., G.M. Simpson and J.M. Naylor. 1984. The physiological basis
of seed dormancy in Avena fatua. IV. Alternative respiration and
nitrogenous compounds. Physiol. Plant. 60:234-238.
Alscher-Herman, R. and A.A. Khan. 1980. Polyribosomes and protein
synthesis in after-ripening pear seeds. Physiol. Plant. 48:285-291.
Alscher-Herman, R., M. Musgrave, A.C. Leopold, and A.A. Khan. 1981.
Respiratory changes with stratification of pear seeds. Physiol. Plant.
52:156-160.
Amen, R.D. 1968. A model of seed dormancy. Botanical Review 34:1-31.
Amthor, J.S. 1989. Respiration and crop productivity. 218 pp. SpringerVerlag, New York.
Azcon-Bieto, J.; H. Lambers and D.A. Day. 1983. The effect of
photosynthesis and carbohydrate status on respiratory rates and the
involvement of the alternative pathway in leaf respiration. Plant Physiol.
10:237-245.
Barthe, P. and C. Bulard. 1978. Bound and free abscisic acid levels in dormant
and after-ripened embryos of Pyrus malus L. cv. Golden Delicious. Z.
Pflanzenphysiol. Bd. 90:201-208.
Barton, L.V. 1945. Respiration and germination studies of seeds in moist
storage. Ann. N.Y. Acad. Sci. 46:185-208.
Beinerts, H. 1977. Iron-Sulfur proteins (W. Lovenberg Ed.) Vol 3. pp 61-100.
Acad. Press. New York.
Berthold, D.A. and J.N. Siedow. 1993. Partial purification of the cyanideresistant alternative oxidase of skunk cabbage (Symplocarpus foetidus)
mitochondria. Plant Physiol. 101:113-119.
Bewley, J.D. and M. Black. 1982. Physiology and biochemistry of seeds in
relation to germination. Vol 2: Viability, dormancy, and environmental
control. Springer-Verlag, Berlin, pp. 258-259.
107
Bianco, J., S. Lasserchere, and C. Bulard. 1984. Gibberellins in dormant
embryos of Pyrus ma/us L. cv. Golden Delicious. J. Plant Physiol.
116:185-188.
Bogatek, R. and S. Lewak. 1978. Respiratory processes during apple seed
after-ripening. Acta Physiol. Plant. 1:45-51
Bogatek, R., Dziewanowska, and S. Lewak. 1991. Hydrogen cyanide and
embryonal dormancy in apple seeds. Physiol. Plant. 83:417-421.
Bogatek, R. and S. Lewak. 1988. Effects of cyanide and cold treatment on
sugar catabolism in apple seeds during dormancy removal. Physiol.
Plant. 73:406-411.
Bogatek, R. and S. Lewak. 1991. Cyanide controls enzymes involved in lipid
and sugar catabolism in dormant apple embryos culture. Physiol. Plant.
73:422-426.
Bogatek, R. and A. Rychter. 1984. Respiratory activity of apple seeds during
dormancy removal and germination. Physiol. Veg. 22:181-191.
Bonner, W.D. and P.R. Rich. 1983. p-Quinol:oxygen oxidoreductase, a new
copper oxidase in plant mitochondria. Plant Physiol. 72:1-19.
Borkowska, B. and L.E. Powell. 1982. Abscisic acid relationships in dormancy
of apple buds. Scientia Hort. 18:111-117.
Bouvier-Durand, M., J. Dereuddre, and D. Come. 1981. Ultrastructural
changes in the endoplasmic reticulum during dormancy release of apple
embryos {Pyrus ma/us L.). Planta 151:6-14.
Bouvier-Durand, M., A. Dawidowicz-Grzegorzewska, C. Thevenot, and D.
Come. 1984. Dormancy of apple embryos. Are starch and reserve
protein changes related to dormancy breaking?. Can. J. Bot. 62:23082315.
Bowen, H.H. and G.W. Dickerson. 1978. relationship of endogenous flower
bud abscisic acid to peach chilling requirements, bloom dates, and
applied gibberellic acid. HortScience 13:694-696.
Bradbeer, J.W. 1968. The role of endogenous inhibitors and gibberellins in
dormancy and germination of Cory/us avellana L. seeds. Planta.
78:266-278.
108
Breidenbach, R.W., D.R. Rank, AJ. Fontana, L.D. Hansen, and R.S. Criddle.
1990. Calorimetric determination of tissue responses to thermal
extremes as a function of time and temperature. Thermochim. Acta
172:179-186.
Brown, J.W. 1939. Respiration of Acorns as related to temperature and afterripening. PI. Physiol. 14:621-645.
Chandler, W.H., M.H. Kimball, G.L. Phillip, W.P. Tufts, and G.P. Weldon.
1937. Chilling requirements for opening of buds of deciduous orchard
trees and some other plants in California. Univ of California, Berkeley
Bui. 611.
Chao, L. and D.R. Walker. 1966. Effect of temperature, chemicals and seed
coat on apricot and peach seed germination and growth. Proc. Am.
Soc. Hort. Sci. 88:232-238.
Cole, M.E., T. Solomos, and M. Faust. 1982. Growth and respiration of
dormant flower buds of Pyrus communis and Pyrus calleryana. J. Amer.
Soc. Hort. Sci. 107:226-231.
Come, D. 1970. Les obstacles a la germination. Masson et cie. Paris, pp.
54-80.
Criddle, R.S., R.W. Breidenbach, D.R. Rank, M.S. Hopkin, and L.D. Hansen.
1990. Simultaneous calorimetric and respirometric measurements on
plant tissues. Thermochim. Acta. 172:213-221.
Criddle, R.S., M. Hopkin, E.D. McArthur, and L.D. Hansen. 1992. The
relationship between temperature coefficient of respiration and plant
distribution. American Naturalist. In press.
Criddle, R.S., R.W. Breidenbach, E.A. Lewis, D.J. Eatough, and L.D. Hansen.
1988. Effects of temperature and oxygen depletion on metabolic rates
of tomato and carrot cell cultures and cuttings measured by calorimetry.
Plant, Cell and Environment 11:695-701.
Criddle, R.S., A.J. Fontana, D.R. Rank, D. Paige, L.D. Hansen, and R.W.
Breidenbach. 1991. Simultaneous measurements of metabolic heat rate,
CO2 production, and 02 consumption by microcalorimetry. Annals
Biochem. 194:413-417.
109
Griddle, R.S., L.D. Hansen, R.W. Breidenbach, M.R. Ward, and R.C. Huffaker.
1990. Effects of NaCI on metabolic heat evolution rates by barley roots.
Plant Physiol. 90:53-58.
Cutting, J.G.M., D.K. Strydom, G. Jacobs, D.U. Bellstedt, K.J. Van Der
Merwe, and E.W. Weiler. 1991. Changes in xylem constituents in
response to rest-breaking agents applied to apple before budbreak. J.
Amer. Soc. Hort. Sci. 116(4):680-683.
Davidson, O.W. 1934. Growing trees from 'non-viable' peach seeds. Proc.
Amer. Soc. Hort. Sci.32:308-312.
Dawidowicz-Grzegorzewska, A. 1980. Anatomy, histochemistry and cytology
of dormant and stratified apple embryos. Ill Structural changes during
early development of seedlings in relation to embryonal dormancy. New
Phytol. 87:573-579.
Dawidowicz-Grzegorzewska, A.
and S.
Lewak.
1978.
Anatomy,
histochemistry and cytology of dormant and stratified apple embryos.
I. General observations and changes in the starch content during afterripening of seeds. New Phytol. 81:99-103.
De la Fuente-Burguillo, P., and G. Nicolas. 1977. Appearance of an alternate
pathway cyanide-resistant during germination of seeds of Cicer
arietinum. Plant Physiol. 60:524-527.
Diaz, D.H. and G.C. Martin. 1972. Peach seed dormancy in relation to
endogenous inhibitor and applied growth substances. J. Amer. Soc.
Hort. Sci. 97:651-654.
Dommes, J. 1988. Changes in mRNA complement during the release of
potato microtubers from innate dormancy. Arch. Int. Physiol.
Biochem. 96:1-10.
Donoho, C. and D. Walker. 1957. Effect of gibberellic acid on breaking of rest
period in Elberta peach. Science. 126:1178-1179.
Doorenbos, J. 1953. Review of the literature on dormancy in buds of woody
plants. Meded. Landbouwhogeschool, Wageningen 53:1-24.
Douce, R. 1985. Mitochondria in higher plants: structure, function and
biogenesis. Academic Press. Orlando, pp. 45-156.
110
du Toit, H.J., G. Jacobs, and D.K. Strydon. 1979. Role of the various seed
parts in peach seed dormancy and initial seedling growth. J. Amer. Soc.
Hort. Sci. 104:490-492.
Ducet, G. 1982. Respiratory coupling in potato tuber mitochondria. Effect of
mitochondrial aging. Physiol. Veg. 20:187-199
Dziewanowska, K., I. Niedzwiedz, I. Chodelska, and S. Lewak. 1979a.
Hydrogen cyanide and cyanogenic compounds in seeds. I. Influence of
hydrogen cyanide on germination of apple embryos. Physiol. Veg.
17:297-303.
Dziewanowska, K., I. Niedzwiedz, and S. Lewak. 1979b. Hydrogen cyanide
and cyanogenic compounds in seeds. III. Degradation of cyanogenic
glycosides during apple seed stratification. Physiol. Veg. 17:687-695.
Ehrenshaft, M. and R. Brambl. 1990.
Respiration and mitochondrial
biogenesis in germinating embryos of maize. Plant Physiol. 93:295-304.
Elliot, J.M. and W. Davison. 1975. Energy equivalents of oxygen consumption
in animal energetics. Oecologia. 19:195-201.
Elthon, T.E. and L. Mclntosh. 1987. Identification of the alternative terminal
oxidase of higher plant mitochondria. Proc. Natl. Acad. Sci. U.S.A.
84:8399-8403.
Elthon T.E.; R.L. Nichels and L. Mclntosh. 1989. Monoclonal antibodies to the
alternative oxidase of higher plant mitochondria. Plant Physiol.
89:1311-1317.
Elthon, T.E. and L. Mclntosh. 1986. Characterization and solubilization of the
alternative oxidase of Sauromatum guttatum mitochondria. Plant
Physiol. 82:1-6.
Esashi, Y., Y. Sakai and R. Ushizawa. 1981. Cyanide-sensitive and cyanideresistant respiration in the germination of cocklebur seeds. Plant
Physiol. 67:503-508.
Esashi, Y., H. Komatsu, R. Ushizawa, and Y. Sakai. 1982. Breaking of
secondary dormancy in cocklebur seeds by cyanide and azide in
combination with C2H4 and Oj and their effects on cytochrome and
alternative respiratory pathways. Aust. J. Plant Physiol. 9:97-111.
Ill
Esashi, Y., Y. Ohara, M. Okazaki, and K. Hishinuma. 1979. Control of
cocklebur seeds germination by nitrogenous compounds: nitrite, nitrate,
hydroxylamine, thiourea, azide and cyanide. Plant Cell Physiol. 20:349361.
Esashi, Y., K. Isuzugawa, S. Matsuyama, H. Ashino, and R. Hasegawa.
1991a. Endogenous evolution of HCN during pre-germination periods
in many seeds species. Physiol. Plant. 83:27-33.
Esashi, Y., S. Matsuyama, H. Ashino, M. Ogasawara and R. Hasegawa.
1991b. B- Glucosidase activities and HCN liberation in unimbibed and
imbibed seeds, and the induction of cocklebur seed germination by
cyanogenic glycosides. Physiol. Plant. 83:34-40.
Flemion, F. 1959. Effect of temperature, light and gibberellic acid on stem
elongation and leaf development in physiologically dwarfed seedlings
of peach and rhodotypos. Contrib. Boyce Thompson Inst. 20:57-70.
Flemion, F, and D.S. De Silva. 1960. Bioassay and biochemical studies of
extracts of peach seeds in various stages of dormancy. Contrib. Boyce
Thompson Inst. 20:365-379.
Flemion, F. 1934. Dwarf seedlings from non-after-ripening embryos of peach,
apple and hawthorn. Contrib. Boyce Thompson Inst. 6:205-209.
Flemion, F. 1936. A rapid method for determining the germinative power of
peach seeds. Contrib. Boyce Thompson Inst. 20:57-70.
Flemion, F. and E. Waterbury. 1945. Further studies with dwarf seedlings of
non-after-ripened peach seeds. Contrib. Boyce Thompson Inst. 13:415422.
Flemion, F. and J. Beardow. 1963. Histological studies of physiologically
dwarfed peach seedlings. I. Structure of anomalous leaves. Contrib.
Boyce Thompson Inst. 22:117-131.
Fontana, A.J., L.D. Hansen, R.W. Breidenbach, and R.S. Criddle. 1990.
Microcalorimetric measurements of aerobic cell metabolism in unstirred
cell cultures. Thermochim. Acta 172:105-113.
Frisby, J.W. and S. Seeley. 1993a. Chilling of endodormantpeach propagules.
II. Initial seedling growth. J. Amer. Soc. Hort. Sci. 118:253-257.
112
Frisby, J.W. and S. Seeley. 1993b. Chilling of endodormant peach propagules.
III. Budbreak and subsequent growth of physiologically dwarfed to near
normal seedlings. J. Amer. Soc. Hort. Sci. 118:258-262.
Gardea, A.A., Y.M. Moreno, A.N. Azarenko, P.B. Lombard, and L.S. Daley.
1993. Changes in metabolic properties of grape buds during
development. J. Amer. Soc. Hort. Sci. (Accepted)
Gianfagna, T.J. andS. Rachmiel. 1983. Changes in gibberellin-like activity and
seed germination. Plant Physiol. Suppl. 72:99.
Gianfagna, T.J. andS. Rachmiel. 1986. Changes in gibberellin-like substances
of peach seed during stratification. Physiol. Plant. 66:154-158.
Goldmark, P.J.; J. Curry; C.F. Morris and M.K. Walker-Simmons. 1992.
Cloning and expression of an embryo-specific mRNA up-regulated in
hydrated dormant seeds. Plant Mol. Biol. 19(3):433-441.
Goodwin, T.W. and E.I. Mercer. 1988. Introduction to plant biochemistry.
Second ed. pp. 162-226. Pergamon press. New York.
Goslin, P.G. and J.D. Ross. 1980. Pentose phosphate metabolism during
dormancy breakage of Cory/us avellana L. Planta 148:362-366.
Gui, M.X., X.M. Wang, and W.Y. Huang. 1991. The effect of light and
exogenous gibberellic acid on respiration pathways during germination
of tomato seeds. Physiol. Plant. 81:403-407.
Guy, R.D., J.A. Berry,M.L. Fogel, and T.C. Hoering. 1989. Differential
fractionation of oxygen isotopes by cyanide-resistant and cyanidesensitive respiration in plants. Planta. 177:483-491.
Hackett, D.P., B. Rice and C. Schmid. 1960. The partial dissociation of
phosphorylation from oxidation in plant mitochondria by respiratory
chain inhibitors. J. Biol. Chem. 3235:2140-2144.
Halinska, A. and S. Lewak. 1978. The presence of bound gibberellins in apple
seeds. Bui. Acad. Polonaise des Sci. Biol. Ser. Cl. II, 26:119-121.
Hansen, L.D., E.A. Lewis, D.J. Eatough, D.P. Fowler, and R.S. Criddle. 1989.
Prediction of long term growth rates of larch clones by calorimetric
measurements of metabolic heat rates. Can. J. For. Res. 19:606-611.
113
Hansen, L.D., and R.S. Criddle. 1989. Batch-injection attachment for the Hart
differential scanning calorimeter. Thermochim. Acta 154:81-88.
Harrington, G.T. 1923. Respiration of apple seeds. J. Agric. Res. 23:117-130.
Hart Scientific. 1991. Hart scientific differential scanning calorimeter User
manual, hart Scientific, Pleasant Grove Utah, 81 p.
Hatefi,
Y. and D.L. Stiggall. 1976. Metal-containing
dehydrogenases. Enzymes. 13(pt.c) : 175-297.
flavoprotein
Hendricks, S.B. and R.B. Taylorson. 1972. Promotion of seed germination by
nitrates and cyanides. Nature 237:169-170.
Hendricks, S.B. and R.B. Taylorson. 1975. Breaking of seeds dormancy by
catalase inhibition. Proc. Natl. Acad. Sci. USA. 72:306-309.
Henry, M.H. and E.J. Nyns. 1975. Cyanide-insensitive respiration. An
alternative mitochondrial pathway. Sub Cell Biochem. 4:1-65.
Hofmann, G.E. and S.C. Hand. 1992. Comparison of messenger RNA pools
in active and dormant Artemia franciscana embryos: Evidence for
translational control. J. Exp. Biol. 164:103-116.
Hu, H. and G.A. Couvillon. 1990. Activities of catalase and pentose
phosphate pathway dehydrogenases during dormancy release in
nectarine seed. J. Amer. Soc. Hort. Sci. 115:987-990.
Humpreys, T.E., and W.M. Dugger, Jr. 1957. The effect of 2,4dichlorophenoxyacetic acid on plant pathways of glucose catabolism in
higher plants. Plant Physiol. 32:136-140.
Hundal, P.S. and H.N. Khajuria. 1979. Effect of
germination of different varieties of peach.
49:417-419.
Huq, S. and J.M. Palmer. 1978. Oxidation of
cyanide-insensitive respiratory pathway in
FEBS Lett. 92:317-320
GA3 and thiourea on seed
Indian J. Agric. Sci.
durohydroquinone via the
higher plant mitochondria.
Isaia, A. and C. Bulard. 1978. Relative levels of some bound and free
gibberellins in dormant and after-ripened embryos of Pyrus ma/us cv.
Golden delicious. Z. Pflanzenphysiol. Bd. 9:409-413.
114
Iversen, E., E. Wilhelmsen, and R.S. Criddle. 1989. Calorimetric examination
of cut fresh pineapple metabolism. J. Food Sci. 54(5): 1246-1249.
James, T.W. and M.S. Spencer. 1979. Cyanide-insensitive respiration in pea
cotyledons. Plant Physiol 64:431-434.
Jarvis, B.C.; B. Frankland, and J.H. Cherry. 1968. Increased nucleic acid
synthesis in relation to the breaking of dormancy of hazel seed by
gibberellic acid. Planta 83:257-266.
Jarvis, B.C. and P.R.M. Shannon. 1981. Changes is poly(A) RNA metabolism
in relation to storage and dormancy-breaking. New Phytol. 88(1 ):31 -40.
Johnson, F.H., H. Eyring, and B. Jones. 1974. The theory of rate processes
in biology and medicine, pp. 550. John Willey and Sons. New York.
Kepczynski, J.; R.M. Rudnicki and A.A. Khan. 1977. Ethylene requirement for
germination of partly after-ripened apple embryos. Physiol. Plant.
40:292-295.
Khan, A.A. 1975. Primary, preventive end permissive roles of hormones in
plant systems. Bot. Rev. 41:391-420.
Khan, A.A. 1971. Cytokinins: permissive role in seed germination. Science
171:853-859.
King, R.W. 1976. Abscisic acid in developing wheat grains and its relationship
to grain growth and maturation. Planta 132(1):43-51.
Kovacs, M.I.P., and G.M. Simpson. 1976. Dormancy and enzyme levels in
seeds of wild oats. Phytochemistry 15:455-458.
La Croix, J.L. and A.S. Jaswal. 1967. Metabolic changes in after-ripening
seed of Prunus cerasus. Plant Physiol. 42:479-480.
Lambers, H. 1980. The biological significance of cyanide-resistant respiration
in higher plants. Plant Cell Env. 3:293-302.
Lambers, H and F. Posthumus. 1980. The effect of light intensity and relative
humidity on growth rate and root respiration of Plantago lanceolata and
Zea mays. J. Exp. Bot. 31:1621-1630.
115
Lambowitz, A.M., E.W. Smith, C.W. Slayman.
1972.
Oxidative
phosphorylation in Neurospora mitochondria. Studies on wild-type, poky
and chloramphenicol induced wild type. J. Biol. Chem. 247:4859-4865.
Lammerts, W.E. 1942. Embryo culture an effective technique for shortening
the breeding cycle of deciduous trees and increasing germination of
hybrid seeds. Am. J. Bot. 29:166-171.
Lance, C, M. Chaveau and P. Bizengremel. 1985. The cyanide-resistant
pathway of plant mitochondria. In: Higher plant cell respiration.
Enc. Plant. Phys. New ser. (R. Douce, D.A. Days, and T. ap Rees, eds).
Springer-Verlag Berlin. Vol. 18. pp.201-247.
Lang, G., J.D. Early, G.C. Martin, and R.L. Darnell. 1987. Endo-, Para-, and
Eco-dormancy: Physiological terminology and classification for
dormancy research. HortScience 22:371-377.
Lemberg, R. and J. Barret. 1973. Cytochromes. Acad Press. London. 580p.
Leopold, A.C. and M.E. Musgrave. 1979. Respiratory changes with chilling
injury of soybeans. Plant Physiol. 64:702-705.
Lewak, S. 1981. Regulatory pathways in removal of apple seed dormancy.
Acta Hort. 120:149-159.
Lewak, S., A. Rychter, and B. Zarska-Maciejewska. 1975. Metabolic aspects
of embryonal dormancy in apple seeds. Physiol. Veg. 13:13-22.
Lewak, S. and R.M. Rudnicki. 1977. After-ripening in cold-requiring seeds. In:
The physiology and biochemistry of seed dormancy and germination.
A.A. Khan, ed. Elsevier/North-Holland, Amsterdam, pp. 193-217.
Lewak, S. and B. Bryzek. 1974. The influence of cytokinins on apple embryo
photo-sensitivity and acid phosphatase activity during stratification.
Biol. Plant. 16:334-340.
Lloyd, D. 1974. The mitochondria of microorganisms. Academic Press.
London. 553p.
Loike, J.D., S.C. Silverstein, and J.M. Sturtevant. 1981. Application of
differential scanning microcalorimetry to the study of cellular processes:
Heat production and glucose oxidation of murine macrophages. Proc.
Natl. Acad. Sci. U.S.A. 78(10):5958-5962.
116
Lyr, H. and T. Schewe. 1975. On the mechanism of the cyanide-insensitive
alternative pathway of respiration in fungi and higher plants and the
nature of the alternative terminal oxidase. Acta Biol. Med. Ger.
34:1631-1641.
Macdonald, M.M. and D.J. Osborne. 1988. Synthesis of nucleic acids and
protein in tuber buds of Solanum tuberosum during dormancy and early
sprouting. Physiol. Plant. 73:392-400.
Maciejewska, V., M. Rye, S. Maleszewski, and S. Lewak. 1974. Embryonal
dormancy and the development of photosynthetic activity in apple
seedlings. Physiol. Veg. 12:115-122.
Matsuoka, M. and T. Asahi. 1983. Mechanism of the increase in cytochrome
c oxidase activity in pea cotyledons during seed hydration. Eur. J.
Biochem. 134:223-229.
McNaughton, J.L., and C.T. Mortimer. 1975. Differential scanning
calorimetry. In: I.R.S.; Physical Chemistry Series 2. Vol. 10. pp 1-43.
H.A. Skinner (Ed.). Butterworths, London.
Mehanna, H.T. and G.C. Martin. 1985. Effect of seed coat on peach seed
germination. Scientia Horticulturae 25:247-254.
Mehanna, H.T., G.C. Martin, and C. Nishijima. 1985. Effects of temperature,
chemical treatments and endogenous hormone content on peach seed
germination and subsequent seedling growth. Scientia Horticulturae.
27:63-73.
Mielke, E.A. and F.G. Dennis. 1975. Hormonal control of flower bud
dormancy in sour cherry (Prunus cerasus L.): II. Levels of abscisic acid
and its water soluble complex. J. Amer. Soc. Hort. Sci. 100:287-290.
Mielke, E.A. and F.G. Dennis. 1978. Hormonal control of flower bud
dormancy in sour cherry (Prunus cerasus L.): III. Effects of leaves,
defoliation and temperature on levels of abscisic acid in flower
primordia. J. Amer. Soc. Hort. Sci. 103:446-449.
Mikolajczak, K.L. 1977. Cyanolipids. Prog Chem. Fats Lipids 15:97-130.
Miyamoto, K. and S. Kamisaka. 1990. Effect of gibberellic acid on epicotyl
growth and carbohydrate distribution in derooted Pisum sativum
cuttings with or without cotyledons. Physiologia Plantarum.
80:357-364.
117
Moller, M., A. Berczi, L.H. van der Plas and H. Lambers. 1988. Measurement
of the activity and capacity of the alternative pathway in intact plant
tissues: Identification of problems and possible solutions. Physiol. Plant.
72:642-649.
Montgomery, R., and C.A. Swenson. 1976. Quantitative problems in the
biochemical sciences (2nd edition) pp. 370. W.H. Freeman and Co.
New York.
Moore, A.L. and J.N. Siedow. 1991. The regulation and nature of the cyanideresistant alternative oxidase of plant mitochondria. Biochim. Biophys.
Acta 1059:121-140.
Morris, C.F.; R.J. Andenberg; P.J. Goldmark and M. Walker-Simmons. 1991.
Molecular cloning and expression of abscisic acid responsive genes in
embryos of dormant wheat seeds. Plant Physiol. 95:814-821.
Nawa, Y. and T. Asahi. 1971. Rapid development of mitochondria in pea
cotyledons during the early stage of germination. Plant Physiol.
48:671-674.
Nikolaeva, M.G. 1969. Physiology of deep dormancy in seeds. Israeli program
for scientific translations. Jerusalem, pp. 146-158.
Nobel, P.S. 1991. Physicochemical and Environmental Plant Physiology.
Academic Press. New York. 635 pp.
Olney, H.O. and B.M. Pollock. 1960. Studies of the rest period. II. Nitrogen
and phosphorus changes in embryonic organs of after-ripening cherry
seed. Plant Physiol. 35:970-975.
Ordentlich, A.; R.A. Linzer and I. Raskin. 1991. Alternative respiration and
heat production in plants. Plant Physiol. 97:1545-1550.
Orlando, B. and F.G. Dennis. 1977. Abscisic acid and apple seed dormancy.
J. Amer. Soc. Hort. Sci. 102:633-637.
Pack, D.A. 1921. After-ripening and germination of Juniperus seeds. Bot. Gaz.
71:32-60.
Paiva, E. and H.A. Robitaille. 1978. Breaking bud rest on detached apple
shoots: effects of wounding and ethylene. J. Amer. Soc. Hort. Sci.
103:101-104.
118
Palmer, J.M. 1976. The organization and regulation of the electron transport
in plant mitochondria. Annu. Rev. Plant. Physiol. 27:133-157.
Parish, D.J. and A.C. leopold. 1978. Confounding of alternate respiration by
lipoxygenase activity. Plant Physiol. 62:470-472.
Perino, C, and D. Come. 1991. Physiological and metabolical study of the
germination phases in apple embryo. Seed Sci. & Technol. 19:1-14.
Pienfield, N. J. and H.V. Davies. 1978. Hormonal changes during after-ripening
of Acer platanoides L. seeds. Z. Pflanzenphysiol. 90:171-191.
Pollock, B.M. 1959. Temperature control of physiological dwarfing in peach
seedlings. Nature (London) 183:1687-1688.
Pollock, B.M. and H.O. Olney. 1959. Studies of the rest period. I. Growth,
translocation and respiratory changes in the embryonic organs of the
after-ripening cherry seed. Plant Physiol. 34:131-142.
Pollock, B.M. 1960. Studies of the rest period. Ill Respiratory changes in leaf
primordia of maple buds during chilling. Plant Physiol. 35(6):975-977.
Pollock, B.M. 1962. Temperature control of physiological dwarfing in peach
seedlings. Plant Physiol. 37:190-197.
Powell, P.E. 1987. Hormonal aspects of bud and seed dormancy in temperatezone woody plants. Hortscience 22(5):845-850.
Rank, D.R., R.W. Breidenbach, A.J. Fontana, L.D. Hansen, and R.S. Criddle.
1991. High and low temperature inactivation of tomato.
Planta
185:576-582.
Rhoads, D.M. and L. Mclntosh. 1991. Isolation and characterization of a
complementary DNA clone encoding an alternative oxidase protein of
Sauromatum guttatum (Schot.). Proc. Natl. Acad, Sci. U.S.A.
88:2111-2126.
Rich, P.R. 1978. Quinol oxidation in Arum maculatum mitochondria and its
application to the assay, solubilization and partial purification of the
alternative oxidase. FEBS Lett. 96:252-256.
Rich, P.R., N.K. Wiegand, H. Blum, A.L. Moore, and W.D. Bonner. 1978.
Studies on the mechanism of inhibition of redox enzymes by substituted
hydroxamic acids. Biochim. Biophys. Acta. 525:325-337.
119
Roberts, E.H. 1964a. The distribution of oxidative-reduction enzymes and the
effects of respiratory inhibitors and oxidizing agents on dormancy in rice
seed. Physiol. Plant. 17:14-29.
Roberts, E.H. 1964b. A survey of the effects of chemical treatments on
dormancy in rice seed. Physiol. Plant. 17:30-43.
Roberts, E.H. 1969. Seed dormancy and oxidation processes. In: Dormancy
and survival. Symp. Soc. Exp. Biol. 23:161-192.
Ross, J.D., and J.W. Bradbeer. 1971. Studies in seed dormancy: V. The
content of endogenous gibberellins in seeds of Corylus avellana L.
Planta 100:208-302.
Ross, J.D. 1985. Metabolic aspects of dormancy. In: Seed physiology. Vol 2.
Germination and reserve mobilization. D.R. Murray (Ed.), pp 45-75.
Academic Press. Orlando, FL.
Ross, J.D., and J.W. Bradbeer. 1971. Studies in seed dormancy: VI The
effects of growth retardants on the gibberellin content and germination
of chilled seeds of Corylus avellana L. Planta 100:303-308.
Ross, J.D. 1983. Metabolic control of dormancy breakage in hazel. Plant
Physiol. Suppl. 72:98.
Rouskas, D., J. Hugard, R. Jonard, and P. Villemur. 1980. Contribution a
I'etude de la germination des graines de Pecher (Prunuspersica Batsch.)
cultivar INRAGf305: effets de la benzyl-amino-purine (BAP) et les
gibberellines GA3 et GA4+7 sur la levee de dormance embryonnaire et
('absence des anomalies foliaires observees sur les plantes issues de
graines non stratifiees. C.R. Acad. Sci. Paris, 291:861-864.
Rudnicki, R. 1969. Studies on abscisic acid in apple seeds. Planta 86:63-68.
Rudnicki, R.; M. Saniewski and D.F. Millikan. 1973. The effect of exogenous
plant hormones upon the stratification of apple seeds. Proc. Res. Inst.
Porno. Ser. E. 3:539-552.
Sakajo, S.; N. Minagawa; T. Komiyama and A. Yoshimoto. 1991. Molecular
cloning of a complementary DNA for antimycin A-inducible messenger
RNA and its role in cyanide-resistant respiration in Hansenula anomala.
Biochim. Biophys. Acta. 1090:102-108.
120
Samish, R.M. 1954. Dormancy in woody plants. Annu. Rev. Plant Physiol.
5:183-204.
Schonbaum, G.R., W.D. Bonner, Jr., B.T. Storey, and J.T. Bahr. 1971.
Specific inhibition of the cyanide-insensitive respiratory pathway in
plant mitochondria by hydroxamic acids. Plant Physiol. 47:124-128.
Seigler, D.S. 1981. Cyanogenic glycosides and lipids: Structural types and
distribution. In: Cyanide in biology (B. Vennesland, E.E. Conn, C.J.
Knowles, J. Westley, and F. Wissing, eds.). Acad. Press, London, pp.
133-143.
Sesay, A., C.R. Stewart and R.M. Shibles. 1986. Effects of KCN and
salicylhydroxamic acid on respiration of soybean leaves at different
ages. Plant Physiol. 82:443-447.
Sharma, H.C. and R.N. Singh. 1980. Effect of stratification temperature and
duration on the level of endogenous inhibitor and its relationship with
dormancy in seeds of subtropical peach [Prunuspersica L. Batsch.) cv.
Sharbati. Indian J. Plant Physiol. 23:26-32.
Siedow, J.N. and M.E. Girvin. 1980. Alternative respiratory pathway. Plant
Physiol. 65:669-674.
Siedow, J.N. and D.A. Berthold. 1986. The alternative oxidase. A cyanideresistant respiratory pathway in higher plants. Physiol. Plant. 66:569573.
Simmonds, J.A. and G.M. Simpson. 1971. Increased participation of PPP in
response to after-ripening and gibberellic acid treatment in caryopses
of Avena fatua. Can. J. Bot. 49:1833-1840.
Sinska, I. 1989. Interaction of ethephon with cytokinin and gibberellin during
the removal of apple seed dormancy and germination of embryos. Plant
Science 64:39-44.
Skubatz, H., W. Tang and B. Meeuse. 1993. Oscillatory heat-production in the
male cones of cycads. J. Exp. Bot. 44:489-492.
Skubatz, H. and B. Meeuse. 1993. Energy loss in tissue slices of the
inflorescence of Sauromatum guttatum (Schott.) analyzed by
microcalorimetry. J. Exp. Bot. 44:493-499.
121
Slater, R.J. and J.A. Bryant. 1982. RNA metabolism during breaking of seed
dormancy of fruits of Acer platanoides (Norway maple). Ann. Bot.
50(2):141-149.
Solomos, T. 1977. Cyanide-resistant respiration in higher plants. Ann. Rev.
Plant. Physiol. 28:279-297.
Stewart, C.R.; B.A. Martin; L. Reding and S. Cerwick. 1990b. Seedling
growth, mitochondrial characteristics, and alternative respiratory
capacity of corn genotypes differing in cold tolerance. Plant Physiol.
92:761-766.
Stewart, C.R.; B.A. Martin; L. Reding and S. Cerwick. 1990a. Respiration and
alternative oxidase in corn seedling tissues during germination at
different temperatures. Plant Physiol. 92:755-760.
Stryer, L. 1988. Biochemistry. 3rd edition 1089 pp. W.H. Freeman and Co.
New York.
Taylor, R.J., and E.B. Dumbroff. 1975. Bud, root, and growth regulator
activity in Acer saccharum during the dormant season. Can. J. Bot.
53:321-331.
Theologies, A. and G.G. Laties. 1978. Relative contribution of cytochromemediated and cyanide-resistant electron transport in fresh and aged
potato slices. Plant Physiol. 62:232-237.
Thevenot, C. 1982. Correlations between axis and cotyledons in apple seeds
embryo dormancy. Z. Pflanzenphysiol. 106:13-26.
Tillberg, E. 1984. Levels of endogenous indole-3-acetic acid in achenes of
Rosa rugosa during dormancy release and germination. Plant Physiol.
76:84-87.
Tillberg, E. 1977. Indoleacetic acid levels in Phaseolus, Zea and Pinus during
seed germination. Plant Physiol. 60:317-319.
Tillberg, E. and N.J. Pinfield. 1981. The dynamics of indole-3-acetic acid in
Acer platanoides seeds during stratification and germination. Physiol.
Plant. 53:34-38.
Tissaoui, T. and D. Come. 1973. Levee de dormance de I'embryon de
pommier (Pyrus malus L.) en I'absence d'oxygene et de froid. Planta
111:315-322.
122
Trewavas, A.J. 1982. Growth substances sensitivity: the limiting factor in
plant growth and development. Physiol. Plant. 55:60-72.
Tuan, D.Y.H., and J. Bonner. 1964. Dormancy associated with repression of
genetic activity. Plant Physiol. 39(5):768-772.
Tukey, H.B. 1933. Effect of photoperiod and temperature on growth of
embryo cultured seedlings. Am. J. Bot. 30:707-711.
Tukey, H.B. and R.F. Carlson. 1945. Morphological changes in peach
seedlings following after-ripening treatments of the seeds. Bot. Gaz.
106:431-440.
Tzou, D.; E. Galson, and E. Sondheimer. 1973. The metabolism of hormones
during seed germination and release from dormancy. Plant Physiol.
51:894-897.
Vallance, K.B. 1952. The germination of the seeds of Rhinanthus crista-galli.
Ann. Bot. 16:409.
Vanlerberghe, G.C. and L. Mclntosh. 1992. Coordinate regulation of
cytochrome and alternative pathway respiration in tobacco. Plant.
Physiol. 100:1846-1851.
Vertucci, C.W. 1990. Calorimetric studies of the state of water in seed
tissues. Biophys. J. 58:1463-1471.
Villiers, T.A. and P.P. Wareing. 1960. Interaction of growth inhibitor and a
natural germination stimulator in the dormancy of Fraxinus excelsior.
Nature 185:112-114.
Visser, T. 1954. After-ripening and germination of apple seeds in relation to
the seed coats. Proc. K. Ned. Akad. Wet. 57:175-185.
Visser, T. 1956b. Some observations on respiration and secondary dormancy
in apple seeds. Proc. K. Ned. Akad. Wet. 59:314-324.
Visser, T. 1956a. The role of the seed coats and temperature in after-ripening,
germination and respiration of apple seeds. Proc. K. Ned. Akad. Wet.
59:211-222.
Wang, S.Y., M. Faust, and G.L. Steffens. 1985. Metabolic changes in cherry
flower buds associated with breaking of dormancy in early and late
blooming cultivars. Physiol. Plant. 65:89-94.
123
Wang, D. and J. Beardow. 1968. Thermo-, photo-effect on rosaceous seeds
during germination as expressed in subsequent seedling development.
Contrib. Boyce Thompson Inst. 24:17-23.
Wang, S.Y. and M. Faust. 1987. Metabolic activities during dormancy and
blooming of deciduous fruit trees. Israel J. Bot. 37:227-243.
Wareing, P.P. 1956. Photoperiodism in woody plants. Annu. Rev. Plant
Physiol. 7:191-214.
Westwood, M.N. 1993. Temperate-zone pomology. Physiology and culture.
Third ed. Timber Press Portland, 523 pp.
Wilson, S.B. 1970. Energy conservation associated with cyanide-insensitive
respiration in plant mitochondria. Biochim. Biophys, Acta.
223:383-387.
Wilson, S.B. 1978. Cyanide-insensitive oxidation of ascorbate + NNN'N'tetramethyl-p-phenylene-diamine mixture by mung bean (Phaseolus
aureus) mitochondria. Biochem. J. 176:129-136.
Wilson, S.B. 1980. Energy conservation by the plant mitochondrial cyanideinsensitive oxidase. Some additional evidence. Biochem. J.
190:349-360.
Wilson, S.B. and W.D. Bonner. 1971. Studies of electron transport in dry and
imbibed peanut embryos. Plant Physiol. 48:340-344.
Wokes, F. and S.G. Wilhimont. 1951. The determination of cyanide in seeds.
J. Pharm. Pharmacol. 3:905-917.
Wood, A. and J.W. Bradbeer. 1967. New Phytol. 66:17-26.
Wrzesniewski, W. 1985. Respiration of Mazzard cherry (Prunus av/'um L.)
seeds during cold stratification interrupted several times with a
dormancy inducing warm temperature. Arboretum Konickie
30:351-366.
Yentur, S., and A.C. Leopold. 1976. Respiratory transition during seed
germination. Plant Physiol. 57:274-276.
Young, E. 1990. Changes in respiration rate and energy of activation after
chilling and forcing dormant apple trees. J. Amer. soc. Hort. Sci.
115(5):809-814.
124
Zarska-Maciejewska, B.; I. Sinska; E. Witkowska, and S. Lewak. 1980. Low
temperature, gibberellin and acid lipase activity in removal of apple
dormancy. Physiol. Plant. 48:532-535.
Zarska-Maciejewska, B. and S. Lewak. 1976. The role of lipases in the
removal of dormancy in apple seeds. Planta 132:177-181.
Zigas, R.P. and B.G. Coombe. 1977a. Seedling development in peach, Prunus
persica (L.) Batsch. I. Effects of testas and temperature. Aust. J. Plant.
Physiol. 4:349-358.
Zigas, R.P. and B.G. Coombe. 1977b. Seedling development in peach, Prunus
persica (L.) Batsch. II. Effect of plant growth regulators and their
possible role. Aust. J. Plant. Physiol. 4:359-369.
Zimmerman, R.H.; M. Lieberman, and O.C. Broome. 1977. Inhibitory effect
of a rhyzobitoxine analog on bud growth after release from dormancy.
Plant Physiol. 59:158-160.
Zimmerman, R.H., and M. Faust. 1969. Pear bud metabolism: Seasonal
changes in glucose utilization. Plant Physiol. 44:1273-1276.
APPENDICES
125
Appendix 1.
Analysis of variance for the temperature coefficient of
metabolism (//) of 'Lovell' seeds stratified at 4C for 0 to 12
weeks.
Dependent Variable: 11
DF
48
98
Sum of
Squares
5517.045106
238.151957
146
5755.197064
R-Square
C.V.
Root MSE
U Mean
0.958620
20.22688
1.558885
7.70699456
Source
DF
Type III SS
Mean Square
F Value
Pr > F
TEMP RANGE
WEEKS
TEMP RANGE*UEEKS
6
6
36
4219.435640
258.932809
1038.676657
703.239273
43.155468
28.852129
289.38
17.76
11.87
0.0001
0.0001
0.0001
Source
Model
Error
Corrected Total
Mean
Square
114.938440
2.430122
F Value
47.30
Pr > F
0.0001
126
Appendix 2.
Analysis of variance for the temperature coefficient of
metabolism (//) of 'Nemaguard' seeds stratified at 4C for
0 to 7 weeks.
Dependent Variable: /i
Source
Model
Error
DF
55
105
Sum of
Squares
4770.306114
1293.608579
Corrected Total
160
6063.914693
R-Square
C.V.
Root MSE
U Mean
0.786671
47.80022
3.509997
7.34305696
Source
DF
Type III SS
Mean Square
F Value
Pr > F
TEMP RANGE
WEEKS
TEMP RANGE*UEEKS
6
7
42
4430.447618
65.609866
232.663835
738.407936
9.372838
5.539615
59.94
0.76
0.45
0.0001
0.6213
0.9978
Mean
Square
86.732838
12.320082
F Value
7.04
Pr > F
0.0001
127
Appendix 3.
Analysis of variance for the qmet and CO2 evolution rates and
the qmet/C02 ratio for 'Lovell' cotyledons stratified at 4C for 0
to 12 weeks and treated with 0 or 500 ppp GA3.
Dependent Variable: qme,
Source
Model
Error
DF
9
20
Sum of
Squares
5.70238881
1.71341049
Corrected Total
29
7.41579930
R-Square
C.V.
Root MSE
QMET Mean
0.768951
32.51885
0.292695
0.90007877
DF
Type III SS
Mean Square
F Value
Pr > F
1
4
4
0.22586457
5.13827931
0.33824493
0.22586457
1.28456983
0.08456123
2.64
14.99
0.99
0.1201
0.0001
0.4371
Source
Model
Error
DF
9
20
Sum of
Squares
396.7849828
41.1700347
Mean
Square
44.0872203
2.0585017
F Value
21.42
Pr > F
0.0001
Corrected Total
29
437.9550175
R-Square
C.V.
Root MSE
C02 Mean
0.905995
31.01339
1.434748
4 .62622100
DF
Type III SS
Mean Square
F Value
Pr > F
1
4
4
6.4294866
359.6260873
30.7294089
6.4294866
89.9065218
7.6823522
3.12
43.68
3.73
0.0924
0.0001
0.0200
Mean
Square
49058.0795
4296.9717
F Value
11.42
Pr > F
0.0001
Source
GA3
STRAT TIME
GA3*STRAT TIME
Mean
Square
0.63359876
0.08567052
F Value
7.40
Pr > F
0.0001
Dependent Variable: C02
Source
GA3
STRAT TIME
GA3*STRAT TIME
Dependent Variable: q^/CC^ RATIO
Source
Model
Error
DF
9
20
Sum of
Squares
441522.7154
85939.4347
Corrected Total
29
527462.1501
R-Square
C.V.
Root MSE
RATIO Mean
0.837070
25.04918
65.55129
261.690367
DF
Type III SS
Mean Square
F Value
Pr > F
1
4
4
66842.6578
300799.3191
73880.7385
66842.6578
75199.8298
18470.1846
15.56
17.50
4.30
0.0008
0.0001
0.0114
Source
GA3
STRAT TIME
GA3*STRAT TIME
128
Appendix 4.
Analysis of variance for the qmet and CO2 evolution rates and
the qmet/C02 ratio for 'Lovell' embryos stratified at 4C for 0 to
12 weeks and treated with 0 or 500 ppp GA3.
Dependent Variable: q^,,
Source
Model
Error
DF
9
20
Sum of
Squares
15.70238536
1.66068553
Corrected Total
29
17.36307089
R-Square
C.V.
Root MSE
QMET Mean
0.904355
11.31620
0.288157
2.54640833
DF
Type III SS
Mean Square
F Value
Pr > F
1
4
4
1.10122100
14.25868418
0.34248018
1.10122100
3.56467104
0.08562004
13.26
42.93
1.03
0.0016
0.0001
0.4156
Source
Model
Error
DF
9
20
Sun of
Squares
101.2528706
32.1104846
Mean
Square
11.2503190
1.6055242
F Value
7.01
Pr > F
0.0002
Corrected Total
29
133.3633552
R-Square
C.V.
Root MSE
C02 Mean
0.759226
22.74592
1.267093
5 .57063667
DF
Type III SS
Mean Square
F Value
Pr > F
1
4
4
18.63289068
70.58337282
12.03660710
18.63289068
17.64584321
3.00915178
11.61
10.99
1.87
0.0028
0.0001
0.1544
Mean
Square
52094.8539
2079.7804
F Value
25.05
Pr > F
0.0001
Source
GA3
STRAT TIME
GA3*STRAT TIME
Mean
Square
1.74470948
0.08303428
F Value
21.01
Pr > F
0.0001
Dependent Variable: C02
Source
GA3
STRAT TIME
GA*STRAT TIME
Dependent Variable: qmet/COj RATIO
Source
Model
Error
DF
9
20
Sum of
Squares
468853.6855
41595.6073
Corrected Total
29
510449.2928
R-Square
C.V.
Root MSE
RATIO Mean
0.918512
9.467377
45.60461
481.702700
DF
Type III SS
Mean Square
F Value
Pr > F
1
4
4
84644.4891
283954.3762
100254.8202
84644.4891
70988.5941
25063.7051
40.70
34.13
12.05
0.0001
0.0001
0.0001
Source
GA3
STRAT TIME
GA3*STRAT TIME