Population, reproductive and ecological aspects of the music volute Voluta musica
ASPECTS OF VEGETATIVE AND REPRODUCTIVE …
Transcript of ASPECTS OF VEGETATIVE AND REPRODUCTIVE …
ASPECTS OF VEGETATIVE AND REPRODUCTIVE PHYSIOLOGY
OF DWARF PHARBITIS NIL
HAROLD ADDRICK SIMMONS, B.S., M.S.
A DISSERTATION
IN
BOTANY
Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for
the Degree of
DOCTOR OF PHILOSOPHY
Approved
c Accepxea
December, 1979
' mi
'A/-'* ^ ' ACKNOWLEDGMENTS /^' . ' ^
I am appreciative to Drs. Thomas Brady, Joe Goodin,
Philip Morey and Daniel Krieg for serving on my doctoral
committee and for their aid and constructive criticism
of this research. I also appreciate the initial guidance
of Dr- Jerry Berlin.
Kim Christie, Susan Erwin, Cal Hoffman and Byron
Stephens were most helpful as laboratory assistants. I
thank Phil Keller for his many discussions concerning
this research.
To Dr. Murray Coulter, who guided and directed this
research, I am especially thankful for his investment of
time, interest, and talent.
Most of all, I thank my wife, Ann, for her support
during this work and for typing this manuscript.
11
CONTENTS
ACKNOWLEDGMENTS ii
ABSTRACT iv
LIST OF TABLES vii
LIST OF FIGURES viii
I. INTRODUCTION 1
II. APEX DEVELOPMENT OF DWARF PHARBITIS IN
RESPONSE TO GIBBERELLIN AND PHOTOPERIOD . . 7
Materials and Methods 8
Results 1^
Discussion 2 + III. A COMPARISON OF THE EFFECTS OF PHOTOPERIOD
AND GIBBERELLIN ON THREE DIFFERENT PROCESSES OF GROWTH AND DEVELOPMENT IN PHARBITIS NIL 32
Materials and Methods 33
Results 3^
Discussion 39
IV. SUMMARY ^7
LIST OF REFERENCES ^8
111
ABSTRACT
Developmental and growth responses resulting from
differences in the status of the endogenous rhythm, phyto-
chrome, and gibberellin (GA). were analyzed in Pharbitis
nil. The effects of these three components on floral in
duction, floral development, stem elongation and leaf
expansion were assayed independently to determine if the
action of each regulatory component is the same or different
in growth and development processes. The status of the
endogenous rhythm and/or phytochrome was effected by
application of specific photoperiodic treatments known
to influence these two components differently. In order
to assay effects of both a change in phytochrome status
and light perception during a light sensitive phase of the
endogenous rhythm, responses were analyzed following
exposure of plants to a diurnal (24 hour) short day with
a light break at the eighth hour of darkness. In order to
show the developmental regulation resulting only from
light impingement on the endogenous rhythm, responses
were analyzed following exposure of plants to a bi-
diurnal (48 hour) short day with a light break at the
eighth hour of darkness. This light break is followed
by a long dark period of sufficient length to allow
iv
phytochrome to become innocuous in reference to flowering.
The influence of GA status was evaluated for each of the
photoperiodic conditions above. The effects of GA were
studied by comparing the growth and development of a GA
deficient dwarf (strain Kidachi), to that of the dwarf
treated with exogenous GA.,, and to that of a normal strain
(Violet) which contains abundant endogenous GA.
Normal and dwarf Pharbitis exposed to diurnal long
days and diurnal short days with a light break remained
vegetative due to combined inhibition imposed by the
phytochrome status and light impingement during the photo-
phobe (light sensitive) phase of the endogenous rhythm.
However, plants exposed to diurnal light break cycles
exhibited development of axillary buds which did not occur
in plants subjected to diurnal long days. Application of
GAo caused increased stem elongation, increased leaf area,
and axillary bud development of plants exposed to diurnal
long days or diurnal light break cycles.
Both normal and dwarf Pharbitis subjected to diurnal
or bidiurnal short days were induced to flower. Shoot apex
analysis of dwarf plants exposed to bidiurnal short days
revealed subapical elongation and development of axillary
buds, characteristics found only on GA-treated plants from
other photoperiods. Both exogenous and endogenous GA
V
increased flowering, stem elongation, and leaf area of
plants treated with diurnal or bidiurnal short days.
Applied GA^ was sufficient to overcome the stem growth
deficiency of the dwarf; however, flower production of the
dwarf strain equaled that of the normal only if it re
ceived the extended dark period of a bidiurnal short day
in conjunction with applied GA-. Therefore, the extended
dark period must facilitate synthesis and/or utilization
of some factor which enhances flowering.
Pharbitis exposed to bidiurnal short days with a
light break were induced to flower; however, the level of
flowering was repressed compared to plants that received
short day treatments. The length of darkness following
the light break was sufficient for phytochrome reversion;
therefore, the repression of flowering was due to light
effect during a light sensitive phase of the endogenous
rhythm. GA^ applied in conjunction with bidiurnal light
break cycles increased flowering, stem elongation, and leaf
area, but had no effect on the size or shape of developing
floral primordia.
These data show that the effects of the endogenous
rhythm, phytochrome status, and GA, may be analyzed
separately and in combination, and that these components
affect development of Pharbitis nil in different ways.
vi
LIST OF TABLES
T a b l e Page
1. Summary From the Literature Concerning Effect of Photoperiodic Treatments 5
2. Number of Terminal and Axillary Apical
Meristems Per Shoot Apex 19
3. Relative Area of Shoot Apex 20
4. Area of Vegetative Apical Meristems Per Shoot Apex 21
5- Combined Width of Floral Apical Meristems Per Shoot Apex , 22
6. Width of Terminal Meristem 23
7 . Flowering Responses of Dwarf and Normal Pharbitis Following Seven Cycles of Designated Photoperiodic Treatments . . . . 35
8. Stem Elongation of Dwarf and Normal Pharbitis Following Seven Cycles of Designated Photoperiodic Treatments . . . . 36
9. Relative Leaf Area of Dwarf and Normal Pharbitis Following Seven Cycles of Designated Photoperiodic Treatments . . . . 37
Vll
LIST OF FIGURES
Figure Page
1. Photoperiodic Treatments 10
•2. Percentage Flowering 13
3. Representative Drawings From Control Plants . I6
4. Representative Drawings From GA., Treated-P l a n t s -? 18
5 . M u l t i v a r i a t e Analysis of Developmental Responses 45
V l l l
CHAPTER I
INTRODUCTION
The transition of a plant bud from a vegetative state
to a flowering state remains one of the most intriguing
problems in the study of plant growth and development
(Chailakhyan, 1979). Although an array of environmental
factors have been shown to influence this transition, the
flowering of photoperiodic plants can be controlled by
manipulation of light/dark cycles; consequently, vegetative
and reproductive development may be selectively studied
(Bernier, 1971). Three components affecting regulation
of development were considered in this project: (1)
responses of plants to gibberellic acid; (2) responses
of plants resulting from the status of the photoreceptive
pigment phytochrome; and (3) responses of plants to an
endogenous rhythm which plays a role in the photoperiodic
response (Evans, 1969)-
Responses of plants to applied gibberellic acid
(GA^) are as follows: (1) bolting and flowering in some
long day (LD) rosette plants (Lang, 1957), but not floral
induction of short day (SD) plants under non-inductive
conditions (Jones, 1973); (2) increased stem elongation
by influencing cell elongation, or cell division, or both
(Sachs, 1965); (3) alteration of leaf area (Gray, 1957;
1
2
Humphries, 1958); and, (4) acceleration of leaf development
(Okuda, 1959) Maksmowych et al., 1976). GA., may influence
flowering indirectly by altering stem growth (Lang and
Reinhard, 196I) and enhancing mitotic activity of a meri
stem to render it more responsive to a floral stimulus •
(Ogawa and Zeevaart, I967). Extraction of vegetative and
flowering Pharbitis nil revealed no auxin differences and
hormone application studies with Pharbitis nil indicated
that GA., is more intricately involved in flowering than
other plant hormones (Ogawa and Zeevaart, I967).
Phytochrome, a photoreceptive pigment involved in
developmental regulation, exists in two forms, Pr. and P .
The far-red absorbing form, P., , is the predominant form
resulting from exposure to red or white light. At the
onset of darkness, P disappears as the red-absorbing
form, P , reappears. Reversion of P ^ to P during a
dark period must occur for floral induction to proceed in
SD plants (Hendricks, I963; Vince-Prue, 1975). In the SD
plant Pharbitis nil, this reversion begins about the sixth
hour of darkness and the proportion of P ^ declines rapidly
until the eighth to tenth hour (Evans and King, 1969). A
light interruption (LB) at the eight hour point of the
dark period of a 24 hour SD photoperiodic treatment re
establishes a high PfpiP^ ratio and results in an inhibi
tion of flowering for SD plants (Vince-Prue, 1975) since
3
the length of darkness after the light break is not suffi
cient for phytochrome reversion (Vince-Prue, 1976).
Biinning (1936) first proposed that an endogenous
rhythm was involved in photoperiodic timing and that it
was comprised of two twelve hour alternating phases, one
termed "photophil," or light-loving and the other
"scotophil," or dark-loving (=light sensitive). This
hypothesis has undergone many revisions (Dunning, 1973),
but experimental evidence shows that an endogenous rhythm
plays a major role in the photoperiodic flowering process
for many SD and LD plants (Hillman, 1976). The endogenous
rhythm also plays a role in the regulation of flowering of
Pharbitis (Hamner and Takimoto, 1964). The light period
of a LD extends into the light sensitive phase and a
light flash during the middle of the dark period of a SD
occurs during a light sensitive phase; therefore, when
light is perceived during a light sensitive phase of the
endogenous rhythm, flowering in a SD plant is inhibited
(Hamner and Hoshizaki, 1974).
The inhibition of flowering, which is seen follow
ing exposure of SD plants to a 24 hour short day with a
light break in the middle of the dark period (24 hour LB),
is therefore due to the combined influence of phytochrome
change and light perception during a light sensitive
phase of the endogenous rhythm (Vince-Prue, 1975). In
4
this paper, floral inhibition resulting in plants exposed
to a 24 hour LB treatment, will be termed floral inhibition
due to the effects of both phytochrome and the endogenous
rhythm.
Using bidiurnal (48 hour) photoperiodic treatments,
Hamner and Takimoto (1964) demonstrated that an endogenous
rhythm functions as a time measuring component for floral
induction in Pharbitis. In a SD plant, such as Pharbitis.
a light interruption eight hours after the beginning of
the dark period in a bidiurnal photoperiodic treatment
(48 hour LB) caused the greatest decrease in flowering
(Hamner and Takimoto, 1964). Since the length of darkness
after the light break was of sufficient duration for phyto
chrome reversion (Evans and King, 1969). this inhibition
of flowering was due to light perception during a light
sensitive phase of the endogenous rhythm (Hamner and
Takimoto, 1964). In this paper, floral repression occurring
in plants subjected to a 48 hour LB treatment, will be
termed floral repression due to the effect of the endoge
nous rhythm only. Some of the more pertinent historical
background for choosing each photoperiodic treatment is
summarized in Table 1.
Unfortunately, there is no defined experimental
treatment in which only phytochrome inhibition can be in
voked without involving inhibition due to light perception
5
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The extent of phytochrome inhibition alone can, however,
be extrapolated since the effects of combined inhibition
from both phytochrome and the endogenous rhythm (24 hour
LB treatment) and the effects of inhibition due to the
endogenous rhythm alone (48 hour LB treatment) can be
compared.
This study compares the effects and interactions of
the endogenous rhythm, phytochrome, and gibberellin, on
flower induction and development, stem elongation, and
leaf expansion of dwarf and normal Pharbitis • Light
break treatments given in either diurnal and bidiurnal
photoperiodic cycles have been used to separate the effects
of phytochrome and the endogenous rhythm in these processes
of growth and development. The effects of these treatments
were studied at both the cellular and organismal levels
using plants with and without applied GAo-
CHAPTER II
APEX DEVELOPMENT OF DWARF PHARBITIS IN RESPONSE
TO GIBBERELLIN AND PHOTOPERIOD
Studies of shoot apex responses to photoperiod have
been limited almost exclusively to LD and SD treatments
(Vince-Prue, 1975)- Pharbitis nil is a qualitative SD
plant, remaining vegetative under LDs and flowering
readily under SDs, and this facilitates comparisons
between vegetative and reproductive main shoot apices.
Marushige (1965a, 196512) has compared buds of normal
Pharbitis in successive LDs and SDs, while Bhar and
Radforth (1969) have compared apex development of plants
under LDs to those exposed to a single inductive SD.
Once floral induction occurred, reproductive development
was essentially the same in response to a single 24 hour
SD or a series of SDs (Bhar and Radforth, I969).
Studies detailing shoot apex responses to applied
GA- have also been documented (Sachs, 19651 Cutter, 1971:
Maksymowych et al., 1976). Applied GA- promotes flowering
if applied to Pharbitis before, during, or just after an
inductive long night (Takimoto, 1969).
This study utilizes dwarf Pharbitis for comparisons
of apex development of plants exposed to photoperiodic
treatments which distinguish between the floral inhibition
7
8
due to the effects of both phytochrome and the endogenous
rhythm to floral repression due to the effect of the
endogenous rhythm only. The interaction of these photo
periodic effects and those of GA- was considered by com
paring the buds of dwarf Pharbitis from each photoperiodic
treatment treated with exogenous GA., to those of plants
given the same photoperiods without GA-.
Materials and Methods
Seeds of dwarf Pharbitis were scarified with con
centrated HpSO^ and germinated in a 1:1 mixture of
vermiculite and sterile soil (Takimoto, 1969). The
experimental treatments were carried out in Percival
Growth Chambers with cool white fluorescent tubes supple
mented with 10?5 of the illumination as incandescent light
to provide a minimum illumination of 100 lux (measured as
17-3 - 18.5 |iW cm'^ nm"-*- with an IL Plant Growth Photo
meter at the cotyledonary surface). Seedlings were
grown in continuous light until five days old, then sub
jected to the photoperiodic treatments shown in Figure 1.
The temperature was maintained at 18.5 - 1.5 C for all
experiments (Hamner and Takimoto, 1964).
A preliminary experiment was conducted to determine
the number of cycles of each photoperiodic treatment re
quired to induce maximum flowering in dwarf Pharbitis.
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After each of ten successive cycles of photoperiodic
treatment, ten plants were removed from the growth chambers
and maintained under continuous light. Ten days after each
set of treatments, the flowering response was measured as
the percentage of plants- possessing floral buds (Takimoto,
1969). The number of cycles required to produce maximum
flowering was seven (Figure 2), therefore seven cycles were
used for comparison of control and GA--treated plants.
One lot (10 plants) of each photoperiodic treatment
served as a control. Another lot received 10 }xg of GA-
per plant. This was applied as 2 ^g in 10 p.1 of O.OO^fo
Tween 20 to the cotyledons during the main light period
of each of the first five photoperiodic cycles.
After seven photoperiodic cycles, the plants were
placed in continuous light until harvested. Ten days after
seven repetitive cycles of 24 hour photoperiodic treat
ments, and three days after seven repetitive cycles of
48 hour photoperiodic treatments, shoot apices were har
vested so that plants were chronologically the same age.
The terminal shoot tip of each plant was harvested and
fixed in FAA, dehydrated with ethanol, infiltrated with
xylene, embedded in paraffin, and mounted. Ten ;im sections
were serially mounted and stained with safranin and fast
green (Jensen, 1962). Five to eight repetitions were
made for each lot of plants. Median or near median
sections were selected for analysis.
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A count of the number of terminal and axillary apical
meristems per shoot apex was initially made. In order to
estimate and compare total size of shoot apices from each
treatment, the "cut and weigh" method of Evans (1972) was
used. Outlines of whole apices (50X) were cut from uni
form paper, weighed, and matched against a standard curve
to estimate relative area. Additional analysis included
an area measurement for all apical meristems per shoot apex
for vegetative plants and width measurements for all apical
meristems per shoot apex for flowering plants. A final
assessment of response was made by measuring width of
only terminal meristems.
Results
Representative camera lucida drawings (25X) of shoot
apices for control plants are shown in Figure J, and those
for GA-treated plants are shown in Figure 4. The number
of terminal and axillary apical meristems per shoot apex
of plants from each photoperiod is summarized in Table 2.
The relative area of the shoot apex as calculated by the
"cut and weigh" method is presented in Table 3. Estimated
areas of apical meristems, calculated from height and
width as shown in Figure 3a for vegetative plants, is in
cluded in Table 4. Width comparisons of floral apical
meristems, calculated as shown in Figure 3b, are presented
Figure 3. Representative drawings from control plants of each photoperiodic treatment. All are 25X. The arrows (h=height and w=width) show how area was measured for vegetative development (e.g. 24 Hour LD) and how width was measured for floral development (e.g. 24 Hour SD). See also Table 2 in relation to Figures 3 and 4.
16
Figure 4. Representative drawings from GA3 treated plants of each photoperiodic treatment. Note that axillary bud development is present on all except the plants from 48 Hour LB.
18
24 Hour LD a
24 Hour SD b
48 Hour SD d
48 Hour LB e
19
TABLE 2
NUMBER OF TERMINAL AND AXILLARY APICAL
MERISTEMS PER SHOOT APEX
Photoperiodic
Treatment
24 Hour LD
24 Hour LB
24 Hour SD
48 Hour SD
48 Hour LB
Response
vegetative
vegetative
floral
floral
floral
(Number Shoot
No GA-
1/4*
2/1
2/2
3/6
1/5
2/2
3/6
1/5
of Tips
Meristems/ Examined)
Plus 10 ;ig GA-
3/8
2/2
3/5
3/3
2/2
3/6
1/8
l/4 = 1 apical meristem each visible on 4 shoot apices
examined and 2/l = 2 apical meristems on 1 shoot apex.
20
Photoperiodic
Treatment
24 Hour LD
TABLE 3
RELATIVE AREA OF SHOOT APEX
Area (cm )
Response
vegetative
No GA 1
2840ax
Plus lOjag GA-
4l l2bx
24 Hour LB
24 Hour SD
48 Hour SD
48 Hour LB
vegetative
floral
floral
floral
4348by
8l50cy
9112cy
3790bx
3212abx
4l90bx
2330ax
3900bx
The r e l a t i v e a rea was ca lcu la t ed from cu t t i ng out camera
l u c i d a drawings of shoot apices (50X) and weighing t o com
pare a g a i n s t a s t anda rd . Means followed by the same
l e t t e r i n each v e r t i c a l column ( a - c ) , or in each h o r i z o n t a l
row ( x , y ) , a re not s i g n i f i c a n t l y d i f fe ren t a t the 0.05
l e v e l acco rd ing t o Duncan's New Multiple Range Test
(Dixon, 1971) .
21
TABLE 4
AREA OF VEGETATIVE APICAL MERISTEMS PER SHOOT APEX
Photoperiodic
Treatment
24 Hour LD
24 Hour LB
Response
vegetative
vegetative
No GA-
{)xm )
324ax
352ax
Area
Plus lOjag GA-
(;am )
550ay
520ay
The area of vegetative apical meristems was calculated from
the height and width as shown in Figures 3a and 4a. Means
followed by the same letter in each vertical column (a),
or in each horizontal row (x,y), are not significantly
different at the 0.05 level according to Duncan's New
Multiple Range Test (Dixon, 1971).
22
TABLE 5
COMBINED WIDTH OF FLORAL APICAL MERISTEMS
PER SHOOT APEX
Photoperiodic
Treatment
24 Hour SD
Response
floral
No GA 1
796cy
Width (>im)
Plus iq^g GA-
540bx
48 Hour SD floral 535by 283ax
48 Hour LB floral 348ax 378ax
The width of floral meristems was calculated as shown in
Figure 3b. Means followed by the same letter in each
vertical column (a-c), or in each horizontal row (x,y),
are not significantly different at the 0.05 level according
to Duncan's New Multiple Range Test (Dixon, 1971).
Photoperiodic
Treatment
23
TABLE 6
WIDTH OF TERMINAL MERISTEM
Mean Width ( m)
Response No GA 1
Plus 10;ag GA-
24 Hour LD
24 Hour LB
24 Hour SD
48 Hour SD
48 Hour LB
vegetat
vegetat
floral
floral
floral
ive
ive
248ax
208ax
796 cy
265ay
348bx
228cx
190bx
360dx
153ax
378dx
Means followed by the same letter in each vertical column
(a-d), or in each horizontal row (x,y), are not signifi
cantly different at the 0.05 level according to Duncan's
New Multiple Range Test (Dixon, 1971).
24
in Table 5- Table 6 shows a comparison of width measure
ments for terminal apical meristems only of both vegetative
and flowering plants.
Discussion
Plants subjected to the 24 hour LD treatment remain
vegetative because the dark period was shorter than the
critical dark period (Takimoto, 1969). Lack of floral
primordia development results because phytochrome is pre
dominately P^^ due to insufficient time for dark reversion
to the innocuous P form (Vince-Prue, 1975). Also, the
light period of the 24 hour LD extends into a light
sensitive phase of the endogenous rhythm (Hamner and
Hoshizaki, 1974), resulting in inhibition of floral in
duction. Pharbitis exposed to the 24 hour LB treatment
remain vegetative because the light interruption shifts
phytochrome to a Pf '-Pj ratio which inhibits flowering in
SD plants, and the light break occurs during a light
sensitive phase of the endogenous rhythm, which interrupts
procession toward floral induction (Vince-Prue, 1975)-
The effect of phytochrome status has been reviewed
by Cummings et al. (1965) and Vince-Prue (1976). In the
24 hour LB treatment, the dark period following the light
interruption is not of sufficient length for reversion to
a P„ :P ratio which does not inhibit flowering in Pharbitis
25
(Evans and King, I969). The effect of the endogenous
rhythm has been reviewed by several workers (Hamner and
Takimoto, 1964; Vince-Prue, 1975; Palmer, 1976). Bunning
(1973) and Hamner and Hoshizaki (1974) have presented both
experimental evidence and hypotheses concerning endogenous
rhythms in a 24 hour cycle. Their perspective is that
phytochrome is the photoreceptive pigment, but cannot
account for all experimental results. It is apparent,
therefore, that both of the above photoperiodic components
inhibit floral induction of Pharbitis in a 24 hour LD or
LB treatment.
Even though plants from both the 24 hour LD and LB
treatments remain vegetative, the morphology of the shoot
apices of plants from these treatments is not the same .
All shoot apices of plants exposed to the 24 hour LB
treatment exhibited axillary bud development (Figure 3c);
however, this was not the case for plants maintained under
24 hour LD treatments (Figure 3a). Comparisons of the
number of apical meristems (Table 2) and the relative
area of the shoot apex (Table 3) show this difference
also.
The differences in effect of the 24 hour LD and LB
treatments may be accounted for by considering the
following: (a) time of occurrence of the inhibitory
Pj^:P ratio of phytochrome; or, (b) the possible effect
26
of the light break on endogenous GA levels within the
plant. The first consideration involves the progression
of P j, to P^ and the time of this progression in the photo
periodic cycle. In the 24 hour LD treatment, the period
of d^kness is eight hours, which is not a sufficient
length for phytochrome reversion in Pharbitis (Evans and
King, 1969). Also, the plant is exposed to a single period
of darkness for P^^ reversion to P . In the 24 hour LB
treatment, Pharbitis is exposed to two periods of darkness
in which the Pf^sPj, ratio begins to change, and neither
period is of adequate length for phytochrome conversion
to a ratio which is innocuous to floral primordia develop
ment .
The second consideration involves comparing the
shoot apices from plants of the 24 hour LB treatment
(Figure 3c and Table 2) to those of GA-treated plants from
the 24 hour LD and LB treatments (Compare Figure 3c to 4a
and 4c. See also Table 2). The light break may cause a
breaking of apical dominance in dwarf Pharbitis by
changing endogenous GA levels. The effect of a light
break in the middle of the dark period has been correlated
with an increase in GAs by Chailakhyan and Lozhnikora
(1966). Since dwarf Pharbitis is a GA deficeint mutant
(Ogawa, 1965), the photoperiodic effect is clearly much
more intricate than just an effect on internal GA levels.
27
Development of floral primordia in control plants
receiving the 24 hour SD treatment progresses more rapidly
than in other inductive treatments (Figure 3b vs. 3d and
3e). The shoot apex of these plants enlarges as flowering
occurs (Table 3), as does the terminal apical meristem
(Table 6). The length of darkness of the 24 hour SD is
adequate for phytochrome to change to a predominant status
of P^, and not interfere with flowering (Hendricks, 1963;
Evans and King, 1969). Also, no light is perceived during
a light sensitive phase of the endogenous rhythm (Hamner
and Hoshizaki, 1974; Heide, 1977), and the length of
darkness provides adequate time for synthesis of a floral
stimulus.
Pharbitis grown under 48 hour SD treatments exhibit
subapical elongation and developing axillary buds
(Figure 3d). Comparable subapical elongation is not seen
in any other Pharbitis plants when GA- is absent. The
developing axillary buds of dwarf Pharbitis from 48 hour
SDs are enlarged and distinction between the terminal and
axillary apical meristems is difficult (Figure 3d)' The
extended dark period of the 48 hour SD treatment affects
floral primordia development differently than the 24 hour
SD treatment but similarily to the 24 hour SD treatment
plus GA- (Figure 3d vs. 4b). It is possible that the sub
apical elongation which occurs in plants subjected to 48
28
hour SD treatments, and which occurs without applied GA
is due to: (1) the extended dark period which may favor
GA synthesis and/or utilization and the result would be
similar to exogenous GA application; or. (2) the extended
dark period which may favor synthesis and/or utilization
of another factor which affects apex development in a
manner similar to that of GA. Chemical feeding experi
ments with kaurene and other GA precursors caused no
growth response in Pharbitis nil Kidachi, but GA- changed
the dwarf habit to a pattern similar to normal Pharbitis
(Katsumi et al., 1964). Extracts of GA-like compounds
from dwarf Pharbitis show little or no GA activity in
bio-assays (Ogawa, 1965; Barendse and Lang, 1972). Also,
no GAs could be detected and confirmed in dwarf Pharbitis
by use of gas chromatography or mass spectrometry (Simmons,
1974). These facts seem to rule out the first interpre
tation.
The results obtained with Pharbitis grown under a
48 hour LB treatment should be compared to those of the
24 hour LB treatment. Plants exposed to the 48 hour LB
treatment produce floral primordia whereas those sub
jected to the 24 hour treatment remain vegetative. A
light interruption eight hours af-er the onset of dark
ness in a bidiurnal cycle (48 hour L3) re-establishes a
high P_..:P ratio but, in Fharbi-Js, the subsequent dark
29
period exceeds the eight to ten hours needed for phyto
chrome to revert to P^ (Evans and King, 1969) and the
P^^:Pj^ ratio is not inhibitory to flowering (Figure 2).
However, the development of floral primordia of Pharbitis
•receiving the 48 hour LB treatment is repressed to a level
below that of plants subjected to SDs. Since the dark
period is of sufficient length for phytochrome reversion,
the effec- on floral development is therefore due to light
perception during a light sensitive phase of the endogenous
rhythm. The developmental regulation effected by both
phytochrome and the endogenous rhythm and that effected
by the endogenous rhythm only should not be considered
the same for ?harbi~is, since responses to the 24 hour LB
and ^3 hour LB treatments are not the game.
Applied .:-AT did not induce flowering of dwarf
Fharbj-js expose! to 24 hour LD, or L3 treatments, in
dicating no direct effect on floral induction. The GA
effect on flowering, for dwarf Pharbitis (and possibly
for other SD plants), must be enhancement of growth and
development of floral buds after induction in "us. GA
accelerated flora- development cut did not affect in-
du:tion in the SD plants (Perilla ocimoides (Horavka
et ai., 19c2) and Cosmos bioinnatus (Molder and Owens,
19^^).
Apices of dwarf Pharbitis, gro^^. in 2'- hour LD or
L3 treatments, show subapical elongation in response to
30
applied GA^ (Figure 4a and 4c). Also, the area of the
apical meristems increases compared to control plants
(Table 4). This is a response which has been documented
as a general characteristic of a GA-treated plant (Sachs,
1965). . Comparison of apices from untreated and GA--treated
plants in 24 hour LDs reveals that, in addition to sub
apical elongation, GA- positively stimulated axillary bud
development. Reference to Figures 3 and 4 clearly shows
that applied GA- affects apical dominance in dwarf
Pharbitis except when grown under conditions of the 48
hour LB treatment. Jacobs and Case (I965) have also
shown that GA- stimulates axillary bud growth and may
play a major role in apical dominance. The floral
primordia of GA-treated plants exposed to the 24 hour SD
treatment are retarded in development compared to non-GA
plants (Figure 4b vs. 3b). For dwarf Pharbitis, GA- re
duces terminal and axillary apical meristem size (Tables
5 and 6 ) but apparently promotes differentiation of
axillary buds into flowers for plants exposed to the 24
hour SD. Dwarf Pharbitis responds to 48 hour SDs and
GA- by producing a floral apex which is greatly reduced
in size compared to those other induced plants (Figure
4d and Tables 5 and 6). No effects of applied GA^ on
the size of buds are noted during the 48 hour LB treat
ment (Tables 5 and 6).
31
These data show that floral inhibition effected by
both phytochrome and the endogenous rhythm (responses to
the 24 hour LB treatment) differs distinctly from the
floral repression evoked by the endogenous rhythm only
(responses to the 48 hour LB treatment). The effect of
GA- on apex development of dwarf Pharbitis also differs
from the above photoperiodic components. The influences
of GA-, the status of phytochrome, and/or the endogenous
rhythm apparently act separately in their effects on
development of the shoot apex.
CHAPTER III
A COMPARISON OF THE EFFECTS OF
PHOTOPERIOD AND GIBBERELLIN ON THREE DIFFERENT
PROCESSES OF GROWTH AND DEVELOPMENT IN PHARBITIS NIL
Pharbitis n U strain Violet (=normal) is a twining
vine with elongated internodes and contains many GA-like
compounds (Ogawa, 1965; Barendse and Lang, 1972).
Pharbitis nil Kidachi is a GA deficient dwarf variety
(Barendse and Lang, 1972). and the dwarf growth habit can
be overcome by GA- application (Takimoto, I969). Both
strains are qualitative SD plants; therefore, vegetative
and reproductive growth development can be analyzed
separately by manipulation of photoperiodic treatments.
In Chapter II. flower induction and development was
emphasized. In the following study, flowering, measured
as the number of floral buds per plant, was analyzed to
represent a change from one developmental mode to an
other; that is. from vegetative to reproductive. Stem
elongation was analyzed to represent a definitive process
of growth, as length, and leaf expansion was analyzed to
represent a process involving growth as a change in size.
These three responses were assayed following
exposure of plants to specific photoperiodic treatments.
The effect of GA- was analyzed by comparing the responses
32
33
of dwarf Pharbitis. normal Pharbitis. and the dwarf
supplemented with exogenous GA^, in combination with the
photoperiodic treatments used to distin^ish the develop
mental regulation of both phytochrome and the endogenous
rhythm from that of the endogenous rhythm only.
Materials and Methods
Procedures from Chapter II were followed for seed
germination and seedling establishment of normal and dwarf
Pharbitis • 'Nt.er. the seedlings were five days old, e;':peri-
aentation began.
To test both the effect of GA- and photoperiod,
twenty plants of dwarf Pharbitis and ten plants of the
normal strain were used for each photoperiodic treatment
shown in Figure 1. Ten of the dwarf plants from each
photoperiod were treated with 10 jag GA- each. This was
applied as 2 g in 10^1 of 0.05?5 Tween 20 to the
cotyledons during the main light period of each of the
first five photoperiodic cycles. The remaining ten dwarf
plants 01 each photoperiod were used as untreated controls.
In order to assay plants which were chronologically
the same age, the growth and development responses were
neasured ten days after seven repetitive cycles of 24
hour photoperiodic treatments (Takimoto, 1969). and three
days after seven repetitive cycles of 48 hour photoperiodic
treatments. Flowering was measured as the number of florae
34
buds per plant (Takimoto. 1969). Stem elongation was
measured as length of the main stem in centimeters. Leaf
expansion was estimated by measuring the length and width
of leaves, and the product of these measurements was ex
pressed as relative leaf area per plant. This estimation
of relative leaf area has been used for symmetrical leaves
by several workers (Manivel and Weaver, 1974; Wargo, 1978)
and has a direct functional relationship to actual leaf
area.
Results
The flowering responses of the dwarf and normal
strains, which result after seven cycles of photoperiodic
treatment, are presented in Table 7. The number of flowers
per plant was the same for plants exposed to either the 24
hour SD or 48 hour SD treatment. Plants subjected to 24
hour LD or 24 hour LB treatments remained vegetative,
while those receiving 48 hour LB treatments produced
about one-third the number of flowers as plants grown in
SDs.
Stem elongation responses, resulting from the
various photoperiodic treatments and/or GA availability,
are shown in Table 8. The photoperiodic treatments seem
to have little effect on stem elongation of dwarf plants
that are GA deficient. However, there is a detectable
35
TABLE 7
FLOWERING RESPONSES OF DWARF AND NORMAL PHARBITIS
FOLLOWING SEVEN CYCLES OF DESIGNATED
PHOTOPERIODIC TREATMENTS
Photoperiodic
Treatment Dwarf
24 Hour LD O.Oax
Flowers per plant
Dwarf + GA,
O.Oax
Normal
O.Oax
24 Hour SD
24 Hour LB
48 Hour SD
48 Hour LB
3.6cx
O.Oax
3.6cx
l.lbx
4.7cy
O.Oax
6.5dy
2.6by
6 .2cz
O.Oax
7.0dy
3.2by
Means followed by the same letter in each vertical column
(a-d), or in each horizontal row (x-z), are not signifi
cantly different at the 0.05 level according to Duncan's
New Multiple Range Test (Dixon, 1971).
36
TABLE 8
STEM ELONGATION OF DWARF AND NORMAL PHARBITIS
FOLLOWING SEVEN CYCLES OF DESIGNATED
PHOTOPERIODIC TREATMENTS
24 Hour SD
24 Hour LB
5•labx
5•labx
Photoperiodic
Treatment Dwarf
24 Hour LD 5.5bx
Length (cm)
Dwarf + GA 1
17.8ay
13.2ay
14. lay
Normal
9-5hz
7.9az
8.7bz
48 Hour SD 5.0ax 12.7ay 7.8az
48 Hour LB 5.4abx 13.7az 11.5CZ
Means followed by the same letter in each vertical column
(a-d), or in each horizontal row (x-z), are not signifi
cantly different at the 0.05 level according to Duncan's
New Multiple Range Test (Dixon, 1971).
37
TABLE 9
RELATIVE LEAF AREA OF DWARF AND NORMAL PHARBITIS
FOLLOWING SEVEN CYCLES OF DESIGNATED
PHOTOPERIODIC TREATMENTS
Photoperiodic
Treatment Dwarf
24 Hour LD 23.3bx
Relative Area (cm )
Dwarf + GA 1
32.Oby
Normal
33.2ay
24 Hour SD 20.6ax 28.lay 43.5bz
24 Hour LB
48 Hour SD
48 Hour LB
26.Obex
26.9CX
33.7dx
32.7by
30.7axy
43.0cy
34.5ay
35.8aby
52.8cz
Means followed by the same letter in each vertical column
(a-d), or in each horizontal row (x-z), are not signifi
cantly different at the 0.05 level according to Duncan's
New Multiple Range Test (Dixon, 1971).
38
difference between stem growth of plants of the 24 hour LD
and 48 hour SD treatments.
The relative leaf area for plants of both strains is
presented in Table 9- Dwarf plants subjected to the 24
hour SD, and not treated with GA-, have a smaller relative
leaf area than plants of the two other 24 hour cycles.
The results shown indicate that, in general,• plants from
bidiurnal, or 48 hour cycles, exhibit a larger relative
leaf area. Dwarf plants grown under the 48 hour LB treat
ment possess the largest relative leaf area of any group
not treated with GA.
Exogenous GA- applied to the dwarf and endogenous
GA of the normal strain caused an increase in flowering
of plants subjected to inductive photoperiods when com
pared to the untreated dwarf. Even though the availability
of GA increased flowering, only dwarf plants grown under
the 48 hour SD treatment, and treated with GA-, had a
floral response equal to that of the normal strain.
Both exogenous GA- applied to the dwarf and endogenous
GA of the normal caused increased stem length compared to
the untreated dwarf. The availability and effect of GA
on stem elongation is significant for every photoperiodic
treatment and the amount of growth effected by exogenous
application indicated that the level of GA^ treatment was
probably more than optimal and exceeded that of the normal
39
strain. Stem length of the normal strain was shorter for
plants subjected to the 24 hour or 48 hour SD treatments
than length of plants exposed to the 24 hour LD or LB '
treatments .
Application of GA3 to the dwarf, and endogenous GA
of the normal, increased leaf area under all photoperiodic
treatments . The leaf area of the GA-treated dwarf was
equal to that of the normal strain only under 24 hour LD,
24 hour LB, and 48 hour SD photoperiodic conditions. The
extended dark period of 48 hour SDs, in conjunction with
GA^, enhanced flowering, but had no such effect on leaf
expansion. Plants of both strains, if subjected to 48
hour LB treatments, developed a greater leaf area than
plants exposed to other photoperiodic treatments.
Discussion
The flowering of both normal and dwarf Pharbitis
follows the predictions made in Table 1. Floral inhibi
tion effected by both phytochrome and the endogenous
rhythm occurs in plants grown in 24 hour LD or 24 hour LB
treatments. Plants exposed to either 24 hour SD or 48
hour SD treatments flower in response to the lengths of
darkness in which a floral stimulus is present. The
flowering response of both strains subjected to the 48
hour LB treatment is repressed to a level below that of
40
plants exposed to SDs. The repression of flowering due to
light effect on the endogenous rhythm is seen as predicted
in Table 1.
Applied GA^ did not induce flowering of dwarf
Pharbitis exposed to 24 hour LD, or 24 hour LB treatments,
indicating no direct effect on floral induction. The GA
effect on flowering, for these two strains of Pharbitis
(and possibly for other SD plants), must be enhancement of
floral bud development after induction in SDs. GA also
accelerated floral development but did not affect induction
in the SD plants Perilla ocimoides (Horavka et al. , 1962) and
Cosmos bipinnatus (Molder and Owens, 1974). Lang and
Reinhard (196I) originally proposed that GA affected
flowering indirectly by altering stem growth. GA may also
affect mitotic activity of a meristem, rendering it more
responsive to a floral stimulus (Ogawa and Zeevaart, 1967).
Plants subjected to the extended dark period of the
48 hour SD treatment, and having available GA, show the
greatest increase in flowering. The exogenous GA- was
adequate in overcoming the growth deficiency of the dwarf;
however, both the extended dark period and the applied GA^
were required to increase flowering to that of the normal.
Imamura and Takimoto (1955). using normal Pharbitis, ob
served an increase in flowers per plant with an increase in
length of the dark period. An increase in flowering with
41
increased length of a dark period was also noted for Lemna
perpusilla (Shibata and Takimoto, 1975). Since plants sub
jected to 24 hour SD or 48 hour SD treatments received the
same amount of exogenous GA^, the concentration of GA^
should not be considered the limiting factor for plants
exposed to 24 hour SD treatments. Two possibilities may
be proposed: (a) the extended dark period may facilitate
GA enhancement of flowering; or (b) the extended dark
period may allow production of a factor which acts in
conjunction with GA to increase flowering. Dwarf Pharbitis
is a GA deficient mutant (Ogawa, 1965; Simmons, 1974);
therefore, the first possibility is unlikely. The ex
tended dark period must favor synthesis and/or utilization
of a factor which enhances flowering in dwarf Pharbitis.
Chailakhyan (1979) has proposed a bicomponent scheme,
comprised of gibberellins and anthesins, in which both are
required for flowering. If the above dark factor could be
considered anthesin, then Pharbitis nil grown under 48
hour SD treatments should prove to be a rich source of
this floral stimulant.
Both exogenous and endogenous GA increased stem
elongation and the amount of GAo applied to the dwarf
stain was more than adequate to overcome the dwarf growth
habit and stem length exceeded those of the normal. Stem
elongation of the normal strain follows a pattern linked
42
to floral induction. Normal plants exposed to the 24 hour
SD, and flowering, have the shortest stem lengths recorded
for 24 hour cycles. Stem elongation of plants subjected
to the 24 hour LD or 24 hour LB treatments and remaining
vegetative, is greater. Normal plants of 48 hour SD treat
ments produce a greater number of flowers but less stem
elongation than plants exposed to 48 hour LB treatments.
This indicates that when flowering is either not in progress
or repressed, GA may be utilized for stem growth. Okuda
(1959) also found that applied GA- resulted in accelerated
development of leaf primordia and increased stem elongation.
The effects of GAs on stem elongation, especially for
genetic dwarfs, have been well documented (Pelton, 1964;
Phinney and West, I960).
Leaf expansion was enhanced by applied GA-, and leaf
area measured for the GA-treated dwarf equaled that of
normal Pharbitis under photoperiodic conditions of the 24
hour LD and 24 hour LB treatments. This also supports the
above statement that GA is utilized in growth processes
when flowering is not in progress. iJ\/hen induced to flower
by 24 hour SD treatments, plants of dwarf Pharbitis ex
hibit less leaf expansion than vegetative plants, whereas
normal Pharbitis (with endogenously available GA) has a
greater leaf area. An increase in leaf expansion with
floral induction has also been reported for the SD plant
43
Chenopodium (Thomas, 196la), and the LD plants Spinacia
oleracea and Trifolium repens (Thomas, 196lb). Com
parisons of responses of plants to 48 hour cycles reveals
that leaf expansion is enhanced by the 48 hour LB treat
ment. This is in contrast to the decrease in flowering
when compared to the 48 hour SD treatment. Thus, available
GA may be utilized in leaf expansion when flowering is re
pressed.
A graphic summary of data from the three tables,
Figure 5. shows that flowering is affected more than
stem elongation or leaf expansion by available GA. When
flowering, leaf area, and stem elongation are charted
simultaneously, the shift of relationships due to GA is
as follows : dwarf control plants form the more closely
aligned cluster; the dwarf plants supplemented with GA-
show an intermediate cluster; and, the normal plants show
the least alignment of the clusters. It is important to
note that the shift of all three sets of clusters is in
the direction of increased flowering. Photoperiodic
treatments 1 and 3, the 24 hour LD and 24 hour LB treat
ments, respectively are adjacent. These results correlate
with the predictions in Table 1 concerning developmental
regulation effected by both phytochrome and the endogenous
rhythm. Photoperiods 2 and 4 are adjacent, which are the
24 hour SD and 48 hour SD treatments, respectively.
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46
Reference to Table 1 shows that neither phytochrome nor
the endogenous rhythm should inhibit flowering in plants
exposed to these photoperiods. Apparently, the effects of
these components on stem elongation and leaf expansion are
similar for plants grown under these photoperiods. Also,
the uniqueness of the 48 hour LB treatment is depicted in
Figure 5. Photoperiod 5, the 48 hour LB treatment, does
not align with any group. This also fits the prediction
in Table 1 that responses to this photoperiodic treatment
must be interpreted -differently than responses to those
above.
These data indicate that developmental regulation
effected by both phytochrome and the endogenous rhythm
differs from that effected by the endogenous rhythm only.
The effect of both exogenous GA on the dwarf and endogenous
GA in the normal also differs from the above photoperiodic
components. The status of the endogenous rhythm, and/or
phytochrome, and the influences of GA, act separately in
their effects on flowering, stem elongation, and leaf
expansion.
CHAPTER IV
SUMMARY
The i n f l u e n c e s of GA, phytochrome, and the endoge
nous rhythm on flower induction and development, stem
elongat ion, and leaf expansion of Pharbitis n i l have been
analyzed at the ce l lu l a r and organismal l eve l s . Data pre
sented support these main points : (a) developmental r e
gu la t ion due to the endogenous rhytrjr alone can c lear ly be
d is t inguished from tha t due to both phytochrome and the
endogenous rhythm (Table 7 and Figure 5); (b) combined
ef fec ts of both phytochrome and the endogenous rhythm
caused a breaking of apical dominance, and th i s i s a
response usua l ly observed for GA-treated plants (Figure
3c vs. 4a, 4b, and 4c) ; (c) if f loral induction occurs, GA
i s u t i l i z e d for flower development instead of stem and
leaf growth (Tables 7-9; Fig-ure 5); (d) the stem growth
deficiency of the dwarf i s overcome by applied GA , but
both GA and an extended dark period are required for
flowering of the dwarf to equal that of the normal (Table
7) , i nd ica t ing tha t an extended dark period f a c i l i t a t e s
synthesis and/or u t i l i z a t i o n of a factor which enhances
flowering; (e) the influences of Z-A, the s tatus of phy-^o-
chrome, and/or the endogenous rhythm are d i s t i nc t ly dif
ferent in these growth and developmental processes.
47
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Bernier, G. 1971. Structural and mxetabolic changes in the shoot apex in transition to floweri-g. Can. J. Bot. 49:303-819.
Bhar, D. S., and N. W. Radforth. 1969. Vegetative and reproductive development of shoot apices of Pharbitis nil as influenced by photoTDericdism. Can. J. Bot. 47:1403-1406.
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Jumming, G. G., S. B. Hendricks, and H. A. Borthwick-1965. Rhythmic flowering responses and phytochrome changes in a selection of Chenopodium rubruro• Can. J. Bot. 43:825-853-
Cutter, S. :-. 1971. Plant Anatomy. Addison-Wesley Co.
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Evans, G. C. 1972. The quantitative analysis of plant growth. Blackwell Scientific Publications.
48
49
'^"^^MacMilia^l;!- ' ' ' ' ' ^^^ Induction of Flowering.
Evans, L. T., and R_. W. King. 1969. Role of phytochrome m photoperiodic induction of Pharbitis nil Z. Pflanzenphysiol. 60:277-288"! '~
Gray, R. A. 1957. Alteration of leaf size and shape and other changes caused by gibberellins in plants. Am. J. Bot. 44:674-682.
Hamner, K. C , and T. Hoshizaki. 1974. Photoperiodism and circadian rhythms; an hypothesis. BioScience 24:407-4l4.
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Heide, 0. M. 1977. Photoperiodism in higher plants: an interaction of phytochrome and circadian rhythms. Physiol. Plant. 39=25-32.
Hendricks, S. B. 1963. Metabolic control of timing. Science l4l:21-27.
Hillman, W. S. 1976. Biological rhythms and physiological timing. Ann. Rev. Plant Physiol. 27:159-179.
Horavka, B., J. Krekule, and F. Seiflova. 1962. An anatomical study of the effect of gibberellic acid on differentiation of the shoot apex in the species Perilla ocimoides L. in short and long days. Biol. Plant. 4:239-245.
Humphries, E. C. 1958. Effect of gibberellic acid and kinetin on growth of the primary leaf of dwarf bean (Phaseolus vulgaris). Nature 181:1081-1082.
Imamura, S., and A. Takimoto. 1955- Photoperiodic responses in Japanese morning glory, Pharbitis nil Chois., a sensitive short day plant. Bot. Mag. Tokyo 68:235-241.
Jacobs, W. p., and D. B. Case. 1965- Auxin transport, gibberellin and apical dominance. Science 148:1729-1731.
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