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Doctoral Dissertations 1896 - February 2014
1-1-1990
Light and hormonal control of leaf abscission.
Zhongyuan Mao University of Massachusetts Amherst
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LIGHT AND HORMONAL CONTROL OF
LEAF ABSCISSION
A Dissertation Presented
By
ZHONGYUAN MAO
Submitted to the Graduate School of the
University of Massachusetts in partial fulfillment
of the requirements for the degree of
DOCTOR OF PHILOSOPHY
MAY 1990
Department of Plant and Soil Sciences Copyright by Zhongyuan Mao 1990
All Rights Reserved LIGHT AND HORMONAL CONTROL OF
LEAF ABSCISSION
A Dissertation Presented
By
ZHONGYUAN MAO
Approved as to the style and content by
Lyle E. Craker, Department Head Department of Plant and Soil Sciences DEDICATION
To my parents, Fusheng Mao and Yianying Yu, who are still plowing yellow earth with cows, for their love and early education which made me a man of today; and to my wife,
Weidong Xu, whose love and understanding has made every¬ thing possible.
iv ACKNOWLEDGEMENT
My sincere thanks go to Dr. Lyle E. Craker, my friend and major adviser, for his constant encouragement and solid support which made the dissertation work easier. I feel grateful to all the committee members for their invaluable comments and insights, especially for Dr. Philip D. Reid
for his assistance of the technique on assaying cellulase activity.
A special thanks to Ms. Ellen Stutsman for her
laboratory assistance and revising of my drafts. I would
also like to thank the other staff members and graduate
students who are currently working in the Department, who,
although not participated in the dissertation work
directly, extended cooperation of laboratory equipment and
friendship.
V ABSTRACT
LIGHT AND HORMONAL CONTROL OF LEAF ABSCISSION
MAY 1990
ZHONGYUAN MAO, B.S. SHAANXI TEACHERS' UNIVERSITY, CHINA
M.S. NORTHWESTERN UNIVERSITY, CHINA
PH.D. UNIVERSITY OF MASSACHUSETTS, USA
Directed by Professor Lyle E. Craker
The mechanism of light control of leaf abscission was studied in coleus (Coleus blumei Benth.) and mung bean
(Viqna radiata (L.) Wilczek) cuttings by evaluating the roles of lAA, carbohydrate, and cellulase in the process of abscission. Treatment of the cuttings with red light maintained high break strength in the abscission zone in both tested plants, whereas treatment with far-red light
initiated a dramatic increase in abscission. Leaf diffusate collected from red light treated leaves contained
3 times more lAA than that collected from leaves maintained
in the dark or treated with far-red light. Treatment of
the cuttings with 2,3,5-triiodobenzoic acid (TIBA), an
auxin transport inhibitor, eliminated the inhibitory
VI effect of red light on abscission. No significant difference in lAA oxidase activity was detected among the
light treatments. The rate of decarboxylation of lAA in
leaf tissue treated with red light, however, was higher than in the leaves receiving dark or far-red light.
Conversion of tryptophan to lAA was higher in red light- treated leaf blades than in leaves maintained in dark or
treated with far-red light. Treatment of mung bean leaf cuttings with red or far-red light, produced no significant differences in the content of fructose 6-phosphate, glucose
plus fructose, sucrose, total soluble sugars, or starch. A
higher level of glucose 6-phosphate was detected in red
light-treated leaves. Loading the tissue with sucrose, a
sugar reported to affect organ abscission, did not change
the effect of light on abscission. The differences in the
rate of lAA synthesis observed in leaf tissue under various
light treatments is proposed as the controlling mechanism
for light regulation of abscission. Total sugar or starch
content in the leaf blade was not related to leaf
abscission, indicating that photoassimilate in leaf tissue
was not involved in light regulation of abscission. While
cellulase activity in the abscission zone accounts for the
decrease in break strength of abscission zone, the enzyme
per se was not affected by light.
vii TABLE OF CONTENT
DEDICATION.iv
ACKNOWLEDGEMENTS.v
ABSTRACT.vi
LIST OF TABLES.x
LIST OF FIGURES.xii
CHAPTERS
1. INTRODUCTION.1
2. LITERATURE REVIEW.5
2.1 Abscission Anatomy Relating to Biochemistry of Abscisison.5
2.2 Plant Hormones and Abscission.8
2.3 External Factors Acting on Internal Metabolism in the Control of Abscission.9
2.4 Non-hormonal Factor Relating to Abscission.14
3. METHODOLOGY.16
3 . 1 General.16
3.1.1 Plant Materials.16 3.1.2 Light Treatments.19 3.1.3 Measurements of Abscission.21 3.1.4 Statistical Procedure.21
3.2 Evaluation of The Role of Auxin.22
3.2.1 The Effect of Auxin on Leaf Abscission.22 3.2.2 Collection and Assay of lAA.24
3.3 Light Effect on lAA Synthesis and Metabolism...26
Vlll 3.4 The Effect of Light on Cellulase and Carbohydrate.2 9
4. RESULTS.3 3
4.1 Light Quality and Abscission.33
4.2 The Role of Auxin in Abscission.38
4.3 The Role of Light on lAA Synthesis and Metabolism.42
4.4 The Role of Cellulase in Abscission.45
4.5 The Role of Carbohydrate in Abscission.49
5. DISCUSSION.59
5.1 The Role of Auxin in the Light Control of Abscission.59
5.2 The Role of Cellulase And Carbohydrate in Leaf Abscission.66
LITERATURE CITED.71
IX LIST OF TABLES
1. Light sources and filters used for light treatments... 19
2. ANOVA for light effect on break strength in mung bean leaf abscission zones (Figure 3).36
3. ANOVA for light effect on break strength in leaf abscission zones in coleus (Figure 4).36
4. ANOVA for light effect on cellulase activity in coleus abscission zone tissue (Figure 4).37
5. The effect of light and leaf area on break strength of leaf abscission zones in coleus.39
6. The effect of external auxin on petiole abscission.... 40
7. The effect of auxin transport inhibition on light control of leaf abscission in coleus.43
8. The effect of light on lAA content of leaf diffusate assayed by GC-MS.44
9. The effect of light on lAA synthesis from tryptophan in mung bean leaf blades.46
10. The effect of light on lAA oxidase activity in mung bean leaf tissue.47
11. The effect of light on decarboxylation of 1-^^C-IAA in mung bean leaf tissue.48
12. ANOVA for light effect on glucose 6-phosphate content in mung bean leaf blades (Figure 6).54
13. ANOVA for light effect on fructose 6-phosphate content in mung bean leaf blades (Figure 6).54
14. ANOVA for light effect on glucose plus fructose content in mung bean leaf blades (Figure 6).55
15. ANOVA for light effect on sucrose content in mung bean leaf blades (Figure 6).55
X 16. ANOVA for light effect on total soluble sugar content in mung bean leaf blades (Figure 7).57
17. ANOVA for light effect on starch content in mung bean leaf blades (Figure 7).57
18. The Effect of exogenous sucrose on mung bean leaf abscission.58
XI LIST OF FIGURES
1. Coleus (A) and mung bean (B) cuttings prepared for the purpose of experiment.18
2. Transmission spectra of light sources and filters used in abscission studies.20
3. The effect of light on break strength in leaf abscission zones of mung bean cuttings.36
4. The effect of light on break strength and cellulase activity in abscission zones of coleus cuttings.38
5. Regression of log lAA concentration and break strength of abscission zones in coleus cuttings.45
6. Light effect on soluble sugar content in mung bean leaf blades.52
7. Light effect on starch and total soluble sugar content in mung bean leaf blades.56
Xll CHAPTER 1
INTRODUCTION
Abscission, the physiological process by which organs such as flowers, fruits, and leaves are released from the parent plant, occurs at a specialized locations known as abscission zones. Abscission zones are composed of several layers of small cells generally located at the proximal end of the associated organ (Jenson and Valdovinos, 1968).
During the abscission process, a separation layer forms inside of the abscission zone and the cells in this layer are activated, enlarge, and actively synthesize and secrete cell wall degrading enzymes (Sexton and Roberts, 1982).
When the abscission process reaches this stage, the attached organ is shed. Generally, plants abscise their organs at maturity or following senescence of the organ.
Unfortunately, plants sometimes shed parts prematurely, resulting in an economic loss to the growers and handlers of many horticultural and agronomic crops.
Many factors are known to influence the abscission process. Investigations on the effect of light have
1 indicated the involvement of phytochrome in abscission, as treatment of tissue with red light can inhibit and treatment with far-red light can promote abscission compared with dark-treated leaves (Curtis, 1978; Decoteau,
1984 ; Craker ^ , 1987). Dark-induced leaf abscission in mung bean (Curtis, 1978, Decoteau and Craker, 1983) and
Coleus (Craker et , 1987), fruit abscission in apples
(Greene ejt aj^. , 1986) , flower and pod abscission in soybeans (Heindl and Brun, 1983; Myers ^ , 1987), and flower abscission in snapdragons (Craker and Decoteau,
1984) have been reported to be regulated by light.
The light receiving site for photocontrol of leaf abscission is apparently located in the leaf blades, since selectively removing or covering sections of leaf tissue reduces the ability of red light to inhibit the abscission process (Decoteau and Craker, 1984). The spatial separation of the light receiving site from the abscission zone suggests the requirement of a translocatable abscission regulator moving from the lighted leaf tissue to the abscission zone for control of the abscission process.
This concept is supported by the observation that illumination of one leaf of the unifoliate (first leaf) pair in mung bean cuttings will inhibit dark-induced abscission of the unlighted leaf (Decoteau and Craker,
2 1984) and by the fact that ethylene production in tissue does not appear to be related to the effect of light on abscission (Decoteau and Craker, 1987). The identity of any light induced endogenous abscission regulator has, however, not been demonstrated.
Phytohormones have been observed to delay or promote abscission and several reviews have summarized their role in the abscission process (Addicott and Lyon, 1973; Jacobs,
1968) . Exogenous application of auxin to plant tissue can control abscission in kidney bean and cotton explants
(Abeles and Rubinstein, 1964; Addicott and Lyon, 1973) and has been commercially successful in controlling abscission in apples, pears, and other fruit crops (Cooper and Henry,
1973; Edgerton, 1973). Although no direct evidence has been available to support the hypothesis, Curtis (1978) has suggested that light regulation of abscission is also under the same hormonal control.
The role of carbohydrates in the abscission process has remained unclear despite studies in several laboratories (Biggs and Leopold, 1957; Martin, 1958;
Streeter and Jeffers, 1979). External application of sucrose to Phaseolus vulgaris explants has been associated with delayed abscission and increased starch deposition in the leaf pulvinus (Martin, 1958) , and sugar is used
3 routinely to enhance adventitious stem abscission of
Impatiens sultani (Wilson et a 1. , 1986 ) . Loss of photoassimilate production also has been suggested as the causal agent in abscission of shaded organs like leaves, flowers, and fruits from plants growing in the field or plants stored in the dark (Force ^ , 1988; Heindl and
Brun, 1983; Schou ^ , 1978). Yet, at irradiances below photosynthetic compensation, red light inhibits and far-red light accelerates dark-induced leaf abscission (Decoteau and Craker,1987; Craker ^ 1987; Myers ^ / 1987).
These results suggest that the effects of light on abscission may be unrelated to photosynthesis or carbohydrate metabolism. The role of carbohydrate in abscission has not been substantiated by direct analysis of sugar levels in the abscising organ.
This study examines the mechanism of light control of leaf abscission and the role of lAA and carbohydrate in abscission.
4 CHAPTER 2
LITERATURE REVIEW
Abscission is the separation of a plant organ from the main body of the plant at the abscission zone, a collection of specific cells located at the base of the abscising organ. The separation of cells in the abscission zone is caused by cell wall degrading enzymes (Addicott and Wiatr,
1977; Berger and Reid, 1979; Sagee ^ ^./ 1980; Sexton and
Roberts, 1982). Some conditions, both internal and external to the plant environment, are known to influence abscission of plant organs (Addicott and Lyon, 1973). The economic as well as theoretical significance of abscission control made abscission an important plant process and the physiology of abscission has been a focus of plant physiology (Webster, 1973; Addicott, 1982; Osborne, 1973;
Sexton and Roberts, 1982).
2.1 Abscission Anatomy Relating to Biochemistry of
Abscission:
The abscission zone consists of several layers of cells, characterized by their smaller size, denser cytoplasm, and highly branched plasmodesmata as compared
5 with neighboring cells (Jenson and Valdovinos, 1968;
Webster, 1973). During the course of abscission 2 to 3 layers of cells (Sexton, 1979) that have strong affinity to polysaccharide dyes (Wilson and Hendershott, 1968) undergo cytological changes. The nuclei enlarge and become conspicuous in the separation layer (Wilson and
Hendershott, 1986). The number of mitochondria may increase or alter the conformation of the matrix density
(Gilliland ^ , 1976). The rough endoplasmic reticulum increases prior to cell wall degradation and during abscission (Jenson and Valdovinos, 1968; Sexton and Hall,
1974). Substantial increases in the number of dictyosomes, the size of the dictyosome cisternae, and the number of dictyosome vesicles appear to be significant aspects of abscission at the subcellar level (Sexton, 1976; Sexton and
Hall, 1974). Small vescicles, considered as being derived from the dictyosomes and containing cell wall degrading enzymes, have been observed to be nearly continuous with the plasmalemma and destined for release into the paramural region. Studies (Sexton, 1976, 1979; Sexton ^ / 1980;
Sexton and Redshaw, 1981) suggest that, before separation, abscission zone cells are active in synthesizing and secreting hydrolytic enzymes responsible for cell wall digestion.
6 Several enzymes are thought to function in cell wall degradation and separation. Pectic enzymes, active in tobacco flower pedicels (Yanger, 1960), cotton leaf petioles (Valdovinos and Muir, 1965), bean petiole explants
(Morre, 1968), citrus petiole explants (Riov, 1974) and citrus pedicels (Greenberg, ^ , 1975), are responsible for the digestion of the middle lamellae. Although Berger and Reid (1979) observed no change in polygalacturonase activity during abscission of Phaseolus vulgaris explants, a radioimmunoassay by Tucker et a 1 . , ( 1980) on a polygalacturonase protein indicated that the enzyme, activated after ethylene treatment, was not synthesized de novo.
Cellulase activity increases dramatically during abscission in several species, appearing after an initial lag phase and before the breakstrength starts to decline
(Craker and Abeles, 1969b; Durbin et ^., 1981). High levels of cellulase accumulate before final fracture (Goren et al.. 1973; Lewis and Varner, 1970; Rasmussen, 1973). Of the several isoenzymes of cellulase observed in the leaf abscission zone of Phaseolus, two were present in large quantities and with high activities (Lewis et a_l. , 1974 ;
Goren and Huberman, 1976; Reid and Strong, 1974) . The
7 isoenzyme, with an isoelectric point (pi) of 4.5, soluble in simple phosphate buffer was not responsible for abscission.
The activity of the other isoenzymes, with pi 9.5, extracted in salt-fortified buffer, increased before the decrease of breakstrength of the abscission zone. Sexton*s immunoassay (Sexton ^ , 1981; Durbin ^ , 1981), and chemical print of Reid ^ al. (1990) have demonstrated nhan the pi 9.5 isoenzyme was not present before lag phase bur was synthesized de novo in the abscission zone.
2.2 Plant Hormones and Abscission
The time course of the abscission process can be divided into a lag phase and a separation phase, according to the responses of abscission zone cells to external hormone treatments. Breakstrength of the abscission zone is often used to identify the beginning of the separation phase (Craker and Abeles, 1969b; Decoteau and Craker.
1983) . Rubinstein and Leopold (1964) indicated that; two stages were present in the lag phase in bean. Stage I. occurring directly after explant excision, is naintainea and extended by auxin and is relatively less sensitive to ethylene (Jackson et a_l. , 1973; Jackson and Osborne, 19 0) .
If auxin application is delayed, the abscission process enters Stage II, in which auxin loses its ability to retara
8 cell separation (Chatterjee and Leopold, 1965; Halliday and
Wangerman, 1972; Hanisch and Bruinsma, 1973). Ethylene or auxin accelerates abscission in Stage II (Jackson and
Osborne, 1970; Chatterjee and Leolpold, 1965). The effect of auxin in Stage II is mediated most probably through elevated ethylene production (Abeles and Rubinstein, 1964;
Morgan and Hall, 1964).
Another hypothesis concerning hormonal control of abscission implies that auxin gradients across the abscission zone determine the rate of abscission (Addicott,
1982) . If the amount of auxin on the distal side of the abscission zone exceeds the amount on the proximal side, abscission will be accelerated. The reversal of the auxin level in the distal and proximal side of abscission zone delays abscission. Although this hypothesis has experimental support, explaining the mechanism by which auxin in the abscission zone functions to regulate abscission is difficult.
2.3 External Factors Acting on Internal Metabolism in the Control of Abscission
Shedding of plant parts generally is associated with environmental conditions, such as temperature, daylength, water and mineral supply, atmospheric gases, pathological
9 agents and physical injury (Addicott and Lyon, 1973). The mechanism by which these external factors affect abscission is still unclear. Plant hormones, functioning as growth and development regulators, also may play a role in abscission.
Among the external environmental conditions, light has a dramatic effect on abscission of leaves (Decoteau and
Craker, 1987, 1984), flowers (Botecelli ^ , 1978), and fruits (Myers ^ , 1987 ; Kadkade ^ , 1979 ; Brooks,
1980). The red portion of the spectrum appears to be the most effective in photocontrol of the abscission process
(Curtis, 1978; Decoteau and Craker, 1983). The red light effect can be reversed by an immediate far-red light
illumination (Decoteau and Craker, 1984). Phytochrome in leaf tissue is believed to be the light receiving pigment.
The mechanism by which phytochrome regulates abscission is unknown, although red light is known to influence the biochemistry and physiology of plants in ways that are
important to abscission. These include altering the levels of carbohydrates and nitrogenous substances (Kasperbauer et a_l. , 1970; Mitrakos and Mantouvalalos, 1972 ; Mitrakos and
Margaris, 1974), respiration, (Hampp and Wellburn, 1979;
Pecket and Al-Charchafchi, 1979), synthesis and activities of enzymes (Penel and Greppin, 1979; Sharma et ad., 1979),
10 and rates of transport of hormones (Rubinstein, 1970;
Goeshl ^ , 1967; Suzuki ^ ad., 1979).
Phytochrome is known to mediate activation or deactivation of several enzymes that may be involved in the light control of abscission (Zucker, 1972; Schopfer, 1977;
Lamb and Lawton, 1983). Peroxidase, an enzyme thought to affect abscission (Hall and Sexton, 1974; Henry, 1979; Van
Lelyveld, 1978) has shown increased activity in far-red light and decreased in activity in red light (Penel and
Greppin, 1979; Penel ^ , 1976). Conversely, Sharma ^ ad., (1976) noticed that red light increased peroxidase activity in leaves. lAA oxidase, an enzyme that degrades lAA, is reported to act similarly to peroxidase and also to be under red light control.
Phytohormones have been observed to delay or promote abscission, and several reviews have summarized their role
in abscission (Addicott and Lyon, 1973; Jacobs, 1968).
Exogenous application of auxin to plant tissue can control abscission in kidney bean and cotton explants (Abeles and
Rubinstein, 1964; Addicott and Lyon. 1973) and has been commercially successful in controlling abscission in apples, pears, and other fruit crops (Cooper and Henry,
1973; Edgerton, 1973). Recently, Decoteau and Craker
(1987) suggested that a diffusible substance similar to
11 indoleacetic acid was produced in the abscising leaf. The production of this hormone, which could be either diffused to the agar block or transported to the opposite leaf petiole was stimulated by red light and inhibited by far- red light. The effect of light on abscission could be transmitted only by this particular hormone. While ethylene could stimulate abscission, its production did not parallel the decrease in break strength of the abscission
zone.
The levels of plant hormones are affected by light
conditions (Muir and Zhu, 1983 ; Reid et a_l. , 1972 ; Suzuki
et al. , 1979) . Therefore, photocontrol of abscission could be associated with regulation of the internal levels or transport of hormones. Gibberellic acid (GA) content was
shown to be influenced by light during seed germination
(Wareing and Thompson, 1976) and dwarf pea epicotyl growth
(Ingram et al. , 1984) . Red light mobilizes stored fat
bodies and increases lipase activity (Smolenska and Lewak,
1974), suggesting GA was released from stored material upon
exposure to red light. Red light has been demonstrated to
induce the release of bound GA and to promote de noyo
synthesis in etiolated wheat leaves (Loveys and Wareing,
1971). Conversely, reduction of GA synthesis or an
12 increase in GA destruction has been suggested in the red light inhibition of dwarf pea growth (Ingram ^ , 1984).
The light effect on hormone levels has also been reported in abscisic acid (Loveys, 1979) , cytokinin (Tucker and
Mansfield, 1972), ethylene (Loveys and Wareing, 1971;
Decoteau and Craker, 1987), and auxin (Bandurski ^ al. .
1977; Tucker, 1976; Muir and Zhu, 1983) in a broad spectrum of plants.
Tryptophan is believed to be the natural precursor of lAA in higher plants. The pool size of tryptophan in plant tissue is usually 3 or more orders higher than the lAA pool and the intermediates from tryptophan to lAA have been identified to be present in plant tissue. The discoveries of easy conversion of tryptophan to lAA by microorganisms
(Schneider and Wightman, 1974) and in non-enzymatic system
(Epstein et a_l. , 1980) caused some doubt as to whether synthesis from tryptophan occurred in higher plants
(Reinecke and Bandurski, 1987) . An enormous body of knowledge suggests that lAA is synthesized from tryptophan
(Wightman and Cohen, 1968; Heerklob and Libbert, 1976;
Sanderberg ^ , 1987).
13 2.4 Non-hormonal Factor Relating to Abscission
High levels of carbohydrates in plant organs have been observed to delay abscission, whereas low levels of carbohydrates in plant organs tend to accelerate abscission
(Addicott, 1982). Results from different laboratories, however, are contradictory. For example, applying sucrose to Phaseolus vulgaris leaf petiole explants delayed abscission of the leaf pulvinus (Martin, 1954) and enhanced starch deposition in this tissue, a factor considered necessary for abscission to occur. Sucrose is used routinely to enhance adventitious stem abscission in
Imoatiens sultani (Wilson ejt aj^. , 1986) . The effect of carbohydrate level on abscission changes with leaf ages and light conditions (Biggs and Leopold, 1957). Stimulative effects were observed in young, fully expanded, and medium aged leaf cuttings at high concentrations of sucrose but only in darkness. Sucrose loses the ability to inhibit abscission under white light especially in aged petiole cuttings, suggesting that photoassimilate masks the sucrose effect, even though the explants were leafless. Lack of carbohydrates also has been suggested as the cause of abscission of lower leaves during shading or dark storage
(Heindl and Brun, 1983 ; Schou ^ ^* / 1978 ; Force ^ ,
1988). At light intensities below the photosynthetic
14 compensation point, red light inhibition of abscission cannot be explained as a photoassimilate limitation on abscission. No direct analysis of carbohydrate in the abscising organs, however , has been done.
15 CHAPTER 3
METHODOLOGY
3.1 General
3.1.1 Plant Materials:
Plant materials used in this study were coleus (Coleus blumei Benth. cv. Ball 2719 Red) and mung bean (Vigna radiata (L.) Wilczek, cv. Jumbo). Coleus was chosen because these plants are easy to grow and are one of the most frequently used plants in studies of abscission,
facilitating comparison of the current research results with the collection of abscission literarure; mung bean plants were chosen because these plants produce uniform seedlings with primary leaves well suited to physiological experiments. Leaf abscission in both of the plant species
has been demonstrated previously to be under light control
(Decoteau and Craker,1987; Craker ^ , 1987).
Coleus plants, propagated by the rooting of shoot
cuttings in sand, were grown in a greenhouse under natural
daylight with a minimum temperature of 18*C in a
loam:peat:sand (1:1:1) mix. The plants were maintained on
a regular pest control and fertilization programs to
16 sustain vigorous growth. Mung beans were grown from seed in vermiculite in a controlled environment chamber with a
16-hour light period (PAR level of 60 mmol m“^s“^) and a temperature of 20 ± 3°C.
For experimental purposes, cuttings were made from both, coleus and mung bean, plants. In coleus, the cuttings were made from leaf and stem tissue of the third or fourth nodes subapical to the apical tip and consisted of the two opposite leaves with approximately 0.5 cm stem above and 2.0 cm of stem below the node (Craker Bt al..
1987) . Except where noted, leaves were trimmed with a razor blade to provide a known leaf area of 1 cm (Figure lA) . The coleus cuttings were maintained in a viable, turgid state during light treatment by submerging the basal portion of the stems in water through holes in a balsa raft floating on the surface of water contained in plastic trays or through holes in parafan film (Parafilm) stretched across the top of 50 cm^ beakers filled with distilled water.
In mung bean, the cuttings, taken from 10-day-old plants, consisted of 5 cm of stem tissue with attached primary opposite leaves and apical bud (Figure IB)
(Decoteau and Craker, 1983). The mung bean cuttings were maintained during light-treatments by immersing the lower
17 -Petiole / ^-Abscission Zone
I Petiole I I Apical Bud Abscission Zone
leaf Blade
Stem
Figure 1. Coleus (A) and mung bean (B) cuttings prepared for the purpose of experiment. Coleus cuttings were prepared from the 3 to 4 node from the apical bud. Mung bean cuttings were prepared from 10 to 12-day-old seedlings.
18 portion of the stem tissue into distilled water contained
in 50 cm beakers and supporting the upper portion of the cutting on a sheet of Parafilm stretched across the top of the beaker.
3.1.1 Light Treatments:
All light treatments were applied in a controlled environment room at 28 ± 2°C, by placing sheets of red or
far-red acetate light filters or an opague layer between
the light source (cool-white fluorescent for red and
incandescent for far-red) and the plant material to provide
red light, far-red light or dark conditions (Decoteau and
Craker, 1983)(Table 1, and Figure 2). Irradiances were 700
mWm“^ and 600 mWm“^ for red and far-red, respectively,
TABLE 1
Light sources and filtersused for light treatments.
Color LiRht source Filter Peak b Band wi
(nm) (nm)
Red 40 W cool white Roscolux ^ 660 50
fluorescent tube Fire #19
Far-red 60 W Incandescent Caloric Amber^ 750 25
bulbs Acrylic #6637
a Roscolux, 36 Bush Avenue, Portchester, NY.
b Local distributor.
19 c o '(/) E a o
Wavelength (nm)
Figure 2. Transmission spectra of light sources and filters used in abscission studies.
20 monitored at the level of the leaf surface with a J6512
irradiance probe on a digital photometer (Model J16,
Tektronix, Inc. Beaverton, OR).
3.1 2 Measurement of TUDscission:
Abscission was measured quantitatively by determining
the force (break strength) necessary to separate the leaf
petiole from the stem at the abscission zone with a
recording abscisor (Craker and Abeles,1969a; Decoteau and
Craker, 1987). Break strength of abscission zones in
freshly prepared cuttings averaged 153g and 17g, for coleus
and mung bean, respectively. Break strength of the
cuttings after treatment was compared with this value and
expressed as percentage. A decrease in break strength
indicates a weakening of the tissue of the abscission zone
and an advancement of the abscission process.
3.1.3 Statistical Procedure;
All experiments were repeated at least three times
with 10 or more plant cuttings per replication, except
where specified. Results were analyzed by analyses of
variance and interpreted by mean separation procedures.
21 3.2 Evaluation of The Role of Auxin
3.2.1 The Effect of Auxin on Leaf Abscission:
Light control of leaf abscission requires the presence of leaf tissue for a constant supply of abscission
inhibitor to the abscission zone. The reserve capacity of the abscission inhibitor in the leaf tissue was evaluated by testing the effect of leaf area on abscission. The coleus cuttings used in this study had leaf areas of 1
(1 X 1, width X length), 2 (1x2), or 6 (2x3) cm^.
These leaf cuttings were maintained in water during the
light treatment. Abscission was measured after 96 hours.
The effectiveness of externally applied auxin in
retarding abscission was studied in bladeless cuttings of
coleus. The cuttings were composed of two opposite
petioles 1 cm long from the abscission zone of each, and
0.5 cm stem above and 1.5 cm stem below the node. A 3 0 mm
drop of 1.5 % agar solution containing the growth regulator
was placed on the distal tip of the bladeless petiole. The
— f\ concentrations of lAA used in this study were 10 ,
5 X 10“^, 10~^, 5 X 10“^, lO”"^, and 5 x 10“"^ mol dm“^,
selected included those the levels demonstrated to be
effective in other physiological processes (Galston and
Baker, 1951). Agar without any growth regulator was used
22 as a control. The bladeless cuttings treated with the growth regulator were placed in a dark environment at temperature of 28 ± 2°C and the break strengths were measured after 2 days.
To test whether transport of auxin from leaf blade to the abscission zone was necessary for the light control of abscission, the movement of auxin from leaf tissue to abscission zone was inhibited by treatment of the leafed coleus cuttings with 2,3,5-triiodobenzoic acid (TIBA), a known inhibitor of auxin transport (Morgan and Durham,
1972; Morris ^ , 1973). Freshly prepared cuttings were either vacuum infiltrated (30 kPa for approximately 30 s)
_ O , , with 5 mol m TIBA water solution using a technique previously described for infiltrating other compounds
(Decoteau and Craker, 1987) or treated by placing a ring of lanolin containing 1 % TIBA around the petiole midway between the leaf blade and abscission zone. Control cuttings were infiltrated with water or treated with lanolin without TIBA. The treated cuttings were then placed in darkness or in red or far-red light for 4 days, and the break strength of the abscission zones was measured at the end of the treatment period.
23 3.2.2 Collection and Assay of lAA:
To verify that lAA is the hormone involved in the light control of leaf abscission, leaf diffusate was collected and assayed for lAA content. The leaf diffusate collection was performed under different light treatments with coleus cuttings having a leaf area of 2 cm^ by using a modified agar-diffusion technique similar to that used for estimating hormone production by other plant organs (Jones and Phillips, 1964) . The petioles of each leaf were cut midway between the blade and abscission zone. To limit microbial growth, the lower 1 cm of each petiole was surface sterilized by submerging in a 1% sodium hypochlorite solution for a minimum of 5 minutes. The end of the stem was subsequently recut 1 mm above the previous cut using a sterile razor blade, and the freshly cut surface of the petiole was embedded immediately
O approximately 0.5 cm into 0.5 cm of sterile, 1% agar contained in a silanized, 1.5 cm glass vial. The vials containing agar and leaf tissue were placed subsequently in
red, far-red light or dark treatment for 48 hours in 4.8 dm^ plastic boxes that were flushed continually with
humidified air to prevent the accumulation of ethylene and
to prevent the explants being dehydrated. After 48 hours,
the agar blocks from each light treatment (20 leaf
24 diffusates/sample) were removed from the glass vials and combined.
The combined agar blocks containing leaf diffusate were spiked with 100 ng of ■^'^C-labeled lAA, acidified to pH
2.5 with HCl , and hand-homogenized in 4 cm^ of dichloromethane to extract the lAA. The aqueous phase, extracted four times, was removed by freezing the samples at -20°C. All of the dichloromethane extracts were combined, dried under nitrogen, and redissolved using three rinses of 0.1 cm^ of acetonitrile. The acetonitrile was concentrated to 0.1 cm under nitrogen, and the lAA was derivatized by adding 0.1 cm^ of N-methvl-N(tert-butvl dimethyl-silyl)-trifluoroacetamide (MTBSTFA) and letting the sample react at 60°C for 20 minutes. The excess
MTBSTFA was evaporated under nitrogen and 50 cm samples of the derivatized lAA were injected into a GC-MS system
(Model 5985, Hewlett-Packard, Avondale, PA) for detection by selected ion monitoring techniques using a 10-m "Ultra
1" column (Hewlett-Packard, Inc. )with helium as the carrier gas. With each sample, 1 ng of anthracene-dlO was coinjected as a reference sample. The quantity of lAA within the diffusate was established by comparison with the known quantity of ^^C-IAA in each sample. The recovery was calculated to be 48 ± 4%.
25 3.3 Light Effect on IMi Synthesis And lAA Metabolism;
To approach the precise mechanism of light control of lAA level detected at the abscission zone, synthesis and metabolism of the hormone in leaf tissue were tested. lAA synthesis from tryptophan was studied in mung bean cuttings. Cuttings, pretreated with red light, far-red light or dark for 24 hours, were vacuum-infiltrated with 4 mmol 2-^^C-tryptophan (1.85 GBg mmol”^)(Decoteau and
Craker, 1987). The cuttings were rinsed with distilled water for a minimum of 3 times to wash away the radioactivity on the surface of the cuttings. Preliminary experiments indicated that the same amount of radioactivity was infiltrated into all cuttings for all the light treatments. The cuttings were subsequently returned to the light treatments for an additional 4 h, after which the leaf blades were harvested and extracted with 80% acetone. The extracts were vacuum-condensed to 0.1 to 0.2 cm^, applied to a polysilicic acid gel thin layer sheet, and developed with chloroform, ethyl acetate, and formic acid (5:4:1) (Craker ^ ^. , 1970). The synthesized lAA was located by comparison with co-chromatographed non¬ radioactive lAA. The spots of lAA (R£=0.8) and tryptophan
26 (R^=0.3) were cut from the thin layer and put into scintillation vials containing 10 cm^ scintillation solution (Opti-Fluor, Packard Instrument Company, Downers
Grove, IL) . Radioactivity in each spot was determined with a liquid scintillation counter (Model 3801, Beckman
Instruments, Fullerton, CA).
To test whether red or far-red light has any effect on lAA oxidase, the enzyme assumed to catalyze the oxidative decarboxylation of lAA in plant tissue, lAA oxidase was extracted from mung bean leaf blades and assayed. The lAA oxidase included free form, ionically bound form and covalently bound form in plant tissue (Thomas e_t aj^. ,
1981) . Preliminary results indicated that covalently bound lAA oxidase in mung bean leaf tissue was negligible and measurement of this form could be eliminated to simplify the experimental procedure. The activity extracted in 50 mol m”^ phosphate buffer (pH 4.7) represents the free lAA oxidase. After centrifugation of the extract at 10,000 g for 4 5 min, the activity in the supernatant was assayed to measure the free lAA oxidase activity. The cell debris was washed with ice-cooled water and re-extracted with 50 mol “ 3 m~^ phosphate buffer (pH 4.7) fortified with 1 mol dm
NaCl. Activity extracted in the salt fortified buffer was presumed to be the ionically bound lAA oxidase. The levels
27 of lAA oxidase activity in each fraction were assayed according to the procedure of Sequeira and Mineo (1966).
Absorbance at 247 nm was measured with a double beam spectrophotometer (Model 124D, Coleman Instruments,
Maywood, IL) and continuously recorded. The lAA oxidase activity was calculated from the linear portion of the graph. One unit of lAA oxidase was defined as the change of 1 unit of absorbance at 247 nm in 1 hour.
The rate of decarboxylation of lAA in mung bean cuttings was measured to determine whether light has any effect on the biochemical reaction. Mung bean cuttings were pretreated with red, far-red light, or dark for 24
• — 3 14 hours and subsequently submerged in 4 mmol m 1- C-IAA
(2.04 GBq mmol”^) water solution and vacuum infiltrated into the tissue (Decoteau and Craker, 1987). The cuttings were rinsed with distilled water a minimum of 3 times to wash away the radioactivity left on the surface of the explants. The infiltrated cuttings were then returned to light treatments for an additional 24 h. Humidified air was continuously flushed through the boxes holding the cuttings, with the air exiting the boxes passing through a
_ 3 CO2 trap 0 f 2 0 cm^ 0 f 4 mol dm NaOH. The trap was replaced at 2, 6, 12 , and 24 hours, after vacuum
28 infiltration of l-^'^C-IAA, respectively, A fraction of 1 cm of NaOH solution from the trap was removed and added to a vial containing 10 cm^ scintillation solution with a high buffering capacity (Atomlight, DuPont, Boston,MA) and counted in a liquid scintillation counter (model 3801,
Beckman Instruments, Fullerton, CA) . The ^"^002 released from the tissue, expressed in cpm, was taken as an indication of lAA oxidation in the cuttings.
3.4 The Effect of Light on Cellulase and Carbohydrate
To test whether light has any direct effect on cellulase, coleus cuttings with or without leaf tissue
(bladeless) were used. About 1 mm of abscission zone tissue from each side of the separation layer was removed after 1, 2, 3, and 4 days for leafed cuttings, 2 days for bladeless cuttings. The tissue was homogenized and extracted with 20 mol m~^ phosphate buffer (pH 6.1) fortified with 1 mol dm~^ NaCl . The extract was centrifuged at about 8,000 g for 15 min (International
Centrifuge, International Equipment Inc. Needham Hts,
Mass.). Cellulase activity was assayed in the supernatant viscosimetrically according to procedure of Lewis and
Varner (1970). One portion of the enzyme extract was mixed
29 with 2 portions of 1% carboxymethy 1-cel lulose type 7HF
(Hercules Powder Co., Wilmington, Del.) in 20 mol m“^ phosphate buffer (pH 6.1). Drainage time through a calibrated portion of a 100 mm^ pipette was used as a measure of viscosity and converted into relative cellulase activity according to the procedure of Durbin and Lewis
(1988). The enzyme reaction and viscosity measurement were performed at room temperature (20 ± 4°C).
To study the role of carbohydrate in the process of leaf abscission, soluble sugars and starch were extracted from leaf blades of mung bean cuttings exposed to different light conditions. Carbohydrate level was analyzed enzymatically using the techniques of Leegood and Furbank
(1984). The 2 leaves on each cutting were homogenized in 1 cm^ of ice-cooled 1 mol dm”^ HClO^, and centrifuged at
8,000g for 10 min. The supernatant was collected, and the precipitated solids were washed with 1 cm distilled water and recentrifuged. The supernatant fractions were combined and neutralized to pH 7 with 5 mol dm ^ K2CO3. A 50 mm^ fraction of the neutralized supernatant was treated sequentially with 0.5 units of glucose 6-phosphate dehydrogenase, 0.5 units of phosphoglucose isomerase, 3 units of hexokinase, and 100 units of invertase in the
30 presence of ATP and NADP (enzymes, ATP, and NADP were purchased from Sigma Chemical Company, St. Louis, MO 63178
USA) to determine the levels of glucose 6-phosphate, fructose 6-phosphate, glucose plus fructose, and sucrose, respectively, in the extract. These sugars were considered representative of the pool of total soluble sugars in the leaf tissue.
The centrifuged pellet was washed three times with distilled water, twice with 1.5 cm^ of the same MES buffer, and digested overnight at room temperature (25 ± 5°C) with
0.4 units of amylase and 14 units of amyloglucosidase. The glucose in the digested solution was assayed as above to determine the starch content of leaf tissue.
For some experiments, sugar concentrations in cuttings were increased artificially by the addition of exogenous sucrose at concentrations reported previously to promotmote or inhibit the abscission process, 0.3% and 3.0% respectively
(Biggs and Leopold 1957; Wilson ^ , 1986). Sucrose, at the two experimental concentrations, was vacuum infiltrated into the leaves using a procedure previously described for incorporating other chemicals into mung bean leaf tissue
(Decoteau and Craker, 1987). Following infiltration, the basal ends of the sucrose-treated and control cuttings
31 (infiltrated with distilled water) were placed in beakers containing distilled water. For comparison, the basal ends of a second set of cuttings, not vacuum-infiltrated, were placed in beakers containing a 0.3% sucrose solution; control cuttings were placed in distilled water. All of the cuttings were placed subsequently under light treatments and break strengths were measured after 3 days .
32 CHAPTER 4
RESULTS
4.1 Light Quality and Abscission
Maintaining mung bean cuttings in darkness caused a loss of break strength in the abscission zone beginning after 48 h in the dark. This loss of break strength continued until total abscission occurred. Treatment of the cuttings with far-red light accelerated the abscission process, causing a measurable loss of break strength to occur by 48 h in the light treatment, with abscission being complete by 72 h. Treatment of the cuttings with red light prevented abscission, inhibiting the loss of break strength in abscission zones and maintaining the break strength at the level observed in growing plants (Figure 3).
The same effects of light on leaf abscission were observed in coleus cuttings. Cuttings with a leaf area of
1 cm^ placed in the dark had a low break strength in their abscission zones. Treatment of the cuttings with far-red light stimulated the loss of break strength, and treatment of the cuttings with red light prevented the occurrence of the dark-induced abscission (Figure 4). While the slight increase in break strength of coleus leaf abscission
33 100 34
abscission zones of mung bean cuttings. Cellulose Activity (B /Abscission Zone) o o o o o o o o o o o o
‘ ' I » I > I ' I ■ I ■ Ina I ■ I ■ I.
[f) >^ o
(D E Figure 4. The effect of light on break strength and T cellulase activity in abscission zones of coleus cuttings. o o o o o o o o o o O CD CO h- CD in Tf ro (N
(PltlUl jO %) M:i6u8j]s >)Daja
35 Table 2
ANOVA for light effect on break strength in mung bean leaf abscission zones (Figure 3).
Source df M.S. F Light (L) 2 3544.3 101.5 * * Time (T) 4 4155.0 119.0 * * LT 8 1222.9 35.0 * *
Error 30 34.9 •
** Significant Table 3 ANOVA for light effect on break strength in leaf abscission zones in coleus (Figure 4). Source df M.S. F Light (L) 2 4047.4 74.1 ** Time (T) 3 4600.3 84.2 ** LT 6 1231.5 22.5 ** Error 24 54.6 ** Significant at the 0.01 probability level. 36 Table 4 ANOVA for light effect on cellulase activity in coleus abscission zone tissue (Figure 4). / Source df M.S. F Light (L) 2 8632.7 613.4 ** Time (T) 4 7444.7 528.9 ** LT 8 2712.0 192.7 ** Error 30 14.1 ** Significant at the 0.01 probability level. zones under red light cannot be explained fully, this increase could be due to growth of tissue during the experimental period, making the tissue stronger. The effect of light on the loss of break strength of abscission zones in coleus cuttings were leaf-area dependent (Table 5). The influence of leaf area on break strength can be observed most readily in far-red light treatment, where abscission in coleus cuttings with leaf areas of 2 or 6 cm^ was delayed compared with that of leaf area of 1 cm^ . The effect of leaf area on delaying abscission can also be detected in the dark treatments, where cuttings with a leaf area of 2 cm^ maintained break strengthes close to the initial level. At the leaf areas 37 of 6 cm^, there were no statistical differences in break strength of the abscission zones of cuttings among the light treatments. All petioles abscised by 48 h if blade tissue was removed. Red light could not prevent the abscission and far-red light will not stimulate the abscission process in bladeless petioles. 4.2 The Role of Auxin in Abscission Application of auxin to the distal tip of leafless petioles from coleus effectively inhibited abscission (Table 6). At lAA concentrations above 10~^ mol dm~^, abscission was prevented, whereas at 10 mol dm lAA, abscission proceeded at a rate similar to that of control cuttings treated with plain agar. Application of TIBA, a known auxin transport inhibitor, enhanced abscission in coleus cuttings with a leaf area of 1 cm (Table 7). When the entire cuttings or petioles were treated with TIBA, the ability of red light to inhibit the loss of break strength in the associated abscission zones was lost (Table 8). Although the break strengths of the abscission zones in control cuttings infiltrated only with water or treated only with lanolin were similar to those of untreated cut¬ tings, the break strength of abscission zones in cuttings 38 Table 5 The effect of light and leaf area on break strength of leaf abscission zones in coleus. Leaf Area (cm^) Light Treatment 6 2 1 0^ (% of initial) Red 108 w 112 w 100 w 0 X Dark 104 w 98 w 60 X 0 y Far-Red 100 w 59 X 24 y 0 Z ANOVA Soursce df MS F Light(L) 2 4768.29370 45.00 ** Size(S) 2 4246.30259 40.07 ** LS 4 971.77093 9.17 * Error 18 105.96333 a Means followed by the same letter indicate nonsignificant at the level of P < 0.05 by Duncan's new multiple-range test (DNMRT). b Bladeless petioles were used in this case. Abscission was measured after 48 hours in the relevant light treatment. * ** Significant at the 0.05 and 0.01 probability level, respectively. 39 Table 6 The effect of external auxin on petiole abscission. Auxin Concentration^ Break Strenath (% of initial) Control (no auxin) 22.6 1 X 10“^ 23.6 1 X 10“^ 41.1 5 X 10“^ 53.2 1 X 10""^ 80.2 5 X lO""^ 104.6 Regression model: y = 194.96 + 29.81x ANOVA Source df SS MS F Mean 1 72480.8 72480.8 Regression 1 15020.4 15020.4 18.16 Residual 18 14890.8 827.3 a lAA, at concentrations indicated, was applied to the top of bladeless petioles. Break strength was measured 48 h after placing the cuttings in dark. ** Significant at the 0.01 probability level. 40 strength ofabscissionzonesincoleuscuttings. Figure 5.RegressionofloglAAconcentration and break Break Strength (% of initial) Log lAAconcentration(Mol) 41 treated with red light and TIBA were reduced by more than 70 percent compared with cuttings exposed to red light but no TIBA treatment. The break strength of abscission zones of cuttings treated with far-red light and TIBA was reduced to zero. The quantity of auxin occurring in the diffusate collected from coleus leaves depended upon the light treat¬ ment to which the leaves were exposed (Table 8). Diffusate collected from leaves treated with red light contained over three times as much auxin as diffusate collected from leaves in the dark. 4.3 The Role of Light in IHA Synthesis and Metabolism The conversion of tryptophan to lAA, the biological pathway of lAA synthesis, was significantly greater in red light treated tissue compared to dark- or far-red light- treated mung bean leaf blades (Table 9). About 50% more lAA was produced from tryptophan in leaf blades treated with red light compared with leaf blades received far-red light treatment. lAA oxidase activity in mung bean leaf blades was not changed under any of the light treatments in either the free form or ionically bound form of the enzyme compared with the dark treatment or compared with the enzyme 42 Table 7 The effect of auxin transport inhibition on light control of leaf abscission in coleus Method of Application of TIBA Vacuum-infiltrated^ Lanolin-applied^ -TIBA +TIBA -TIBA +TIBA Light _ (% initial break strength)^ Red light 103 X 15 z 102 X 2 Far-red light 43 X 0 X 50 X ANOVA Source df MS F Light (L) 1 8855.0 51.1 ■k-k TIBA (T) 1 25350.0 146.3 ** Way (W) 1 104.2 0.6 n.s. LT 1 1723.8 10.0 * * LW 1 22.0 0.1 n.s. TW 1 35.5 0.2 n.s. LTW 1 131.6 0.8 n.s. Error 16 173.3 a A 5 mmol m~ ^ TIBA water solution is vacuum infiltrated into the cuttings, b A band of lanolin, containing 1% TIBA, was placed around the treated petiole midway between the distal end and the abscission zone. c Means of the same light treatment followed by the same letter are nonsignificant at the 0.05 probability level by DNMRT. ** Significant at the 0.01 probability level, n.s. Nonsignificant at the 0.05 probability level. 43 Table 8 The effect of light on lAA content of leaf diffusate assayed by GC-MS. Light Treatment Auxin Level^ (ng/sample) Red light 84.8 X Dark 27.5 y Far-Red 17.2 y ANOVA Source df M.S. F Light (L) 2 3606.76806 6.2 * Error 6 582.46365 . 2 a Each sample represents the diffusate from 20 2-cm leaf cuttings for 48 hr, beginning with placement of the tissue in the indicated light treatment. Means followed by the same letter are not significantly different at the 0.05 probability level by DNMRT. * Significant at the 0.05 probability level. 44 activity in fresh cuttings (Table 10) . No significant differences occured among light treatments for lAA oxidase activity 24 h after the initiation of light treatments. Of the lAA oxidase activities, 3/4 were present in the free form and 1/4 were present in the ionically bound form, an insignificant amount of lAA oxidase activity was observed in the covalently bound form. lAA oxidation rate, as indicated by the release of ^^C02 from 1-^^C-IAA, was significantly affected by the treatment of the mung bean cuttings with red or far-red light or dark (Table 11) . The release of ^^C02 from the cuttings was very rapid in the first two h after supplying the tissue with 1-^'^C-IAA, and subseguently proceeds at a much lower rate (about one tenth the initial rate) . The low rate of oxidation was maintained until the end of the experiment, 24 h after 1-^^C-IAA infiltration. The rate of decarboxylation was significantly higher in red light treated cuttings compared with the other light treatments. 4.4 The Role of Cellulase in Abscission Cellulase activity in crude extracts of abscission 2 zone tissue from coleus cuttings with a leaf area of 1 cm depended upon the light treatment. Cellulase activity in 45 Table 9 The effect of light on lAA synthesis from tryptophan in mung bean leaf blades. Light Treatment lAA (cpm)^ Red 85 X Dark 61 y Far-red 54 y ANOVA Source df M.S. F Light(L) 2 1121.7778 7.54 * Error(LR) 6 148.7370 a As measured by conversion of 2-^'^C-tryptophan to ^"^C-IAA; means followed by the same letter are not significantly different at the P < 0.05 level, by DNMRT. * Significant at the 0.05 probability level. 46 Table 10 The effect of light on lAA oxidase activity in mung bean leaf tissue. lAA Oxidase Activity^ Treatment (unit/g FW) Free Bound Initial 38.9 X 9.9 y Red light 34.2 X 12.4 y Dark 39.8 X 11.4 y Far-red light 37.5 X 11.2 y ANOVA Source df MS F Light (L) 2 22.05 0.18 n.s. Form (F) 1 4376.84 35.92 ** LF 2 99.86 0.82 n.s. Error 22 121.86 a Activities were measured at 24 hours after light treatment. Means of the same column followed by the same letter are not significant at the level of P < 0.05 by DNMRT. ** Significant at the 0.01 probability level, n.s. Nonsignificant at the 0.05 probability level. 47 Table 11 The effect of light on decarboxylation of 1-^^c-IAA in mung bean leaf tissue. Treatment Sampling Time Red light Dark Far-red light (hours)^ (cpm / sample / hour) b 2 92.6 X 8 6.6 X 55.6 y 6 16.4 X 17.3 X 15.1 X 12 22.9 X 16.3 xy 14.2 y 24 20.2 X 13.8 xy 12.1 y ANOVA Source df MS F IT) CO Light (L) 2 552.1 • ** Time (T) 3 8572.9 90.9 ** LT 6 246.5 2.6 * Error 24 94.3 a Cuttings were light conditioned for 24 h before vacuum infiltration (0 hour) with l-^'^C-IAA water solution, b Means of the same line followed by the same latter are not significantly different at the level of P < 0.05 by DNMRT. * ** Significant at the 0.05 and 0.01 probability level, respectively. 48 abscission zone of fresh tissue (zero time) from coleus plants was undetectable. Treatment of the cuttings with red light maintained very low cellulase activity in the abscission zone tissue, whereas treatment of the tissue with far-red light or dark caused a sharp increase in cellulase activity in the abscission zone tissue by 48 h after the initiation of the treatment (Figure 4). The appearance of cellulase activity in the abscission zone tissue was slightly earlier than the decrease of break strength of the tissue in dark and far-red light treatments. Induction of cellulase in the abscission zone tissue was enhanced if leaf tissue was removed. Cellulase activity in the abscission zones of leafless petioles reached to maximum in 48 h no matter which light treatment was applied to the cuttings. The relationship of light to cellulase activity was diminished when leafless cuttings were used. 4.5 The Role of Carbohydrate in Abscission: Glucose 6-phosphate, fructose 6-phosphate, and glucose plus fructose levels in leaf blades declined from levels in freshly cut leaves (Figure 6A, B, C), with a sharp decrease in these sugar contents in the first day followed by stabilization at relatively low levels of sugar for the 49 following days. Sucrose content decreased from the levels measured in freshly cut leaves during the 12 to 48 h time period after the initiation of light treatment, and then increased slightly between 48 and 72 h (Figure 6D), the time at which the far-red light treated leaves were abscising. Highly significant differences were observed in glucose 6-phosphate among light treatments. Red light maintained a relatively higher level of glucose 6-phosphate than far-red light or dark treated tissue. No statistical differences among light treatments were obtained for the levels of any other soluble sugars or their combination. Starch levels appeared to decrease slightly from levels in freshly cut leaves, but no significant differences in starch content were detected among light treatments over the course of the experiment (Figure 7) . At the time of abscission, 80% of the starch still remained in the leaf tissue. Total soluble sugar content decreased dramatically under all light treatments during the first 24 h and continued to decrease at a slower rate over the following days. No significant difference in total soluble sugars was observed among leaves treated with red or far-red light or maintained in the dark at any of the sampling times. The low levels of soluble sugar in leaf tissue were maintained throughout the experimental 50 period until 96 h, at which the far-red and dark treated leaves had all abscised (data not presented). Treatment of the mung bean cuttings with exogenously apllied sucrose did not alter the effects of light on abscission (Table 18). Red light still maintained high break strength, while far-red light still enhanced abscission; the presence or absence of sugar had no effect. The method of applying sugar to the cuttings did not affect the results. No significant differences were observed between control and sugar treatments. Highly significant differences, however, were noted in light treatments. 51 Figure 6. Light effect on soluble sugar content in mung bean leaf blades. The mung bean cuttings were prepared from 10 to 12 day old seedlings and placed in red or far- red light, or in darkness. Sugars were extracted from the leaf blades and assayed at the times indicated. 52 Soluble Sugars Time (hours) 53 Table 12 ANOVA for light effect on glucose 6-phosphate content in mung bean leaf blades (Figure 6). Source df MS F Light (L) 2 0.01102 7.03 n.s. Time (T) 4 0.33015 210.67 ** LT 8 0.00226 1.44 n.s. Error 91 0.00157 ** Significant at the 0.01 probability level, n.s. Nonsignificant at the 0.05 probability level. Table 13 ANOVA for light effect on fructose 6-phosphate content in mung bean leaf blades (Figure 6). Source df MS F Light (L) 2 0.00178 0.49 n.s. Time (T) 4 0.07737 21.29 ** LT 8 0.00165 0.46 n.s. Error 82 0.00363 ** Significant at the 0.01 probability level • n.s. Nonsignificant at the 0.05 probability level 54 Table 14 ANOVA for light effect on glucose plus fructose content in mung bean leaf blades(Figure 6). Source df MS F Light (L) 2 0.00943 0.11 n.s. Time (T) 4 0.21256 2.54 * LT 8 0.02145 0.26 n.s. Error 74 0.08369 * Significant at the 0. 05 probability level. n. s. Nonsignificant at the 0.05 probability level. Table 15 ANOVA for light effect on sucrose content in mung bean leaf blades (Figure 6). Source df MS F Light (L) 2 0.00043 0.40 n.s. Time (T) 4 0.00285 2.62 * LT 8 0.00063 0.58 n.s. Error 86 0.00109 * Significant at the 0 .05 probability level • n.s Nonsignificant at the 0.05 probability level. 55 (/) V- □ O JZ Q) E i— Figure 7. Light effect on starch and total soluble sugar content in mung bean leaf blades. (3Uj/:iiun 8soon|6 |oujuj) :^U9:^uoQ ai^DjpAqoqjDQ 56 Table 16 ANOVA for light effect on total soluble sugar content in mung bean leaf blades (Figure 7). Source df MS F Light (L) 2 0.09152 0.68 n.s. Time (T) 4 3.54686 26.26 ** LT 8 0.03547 0.26 n.s. Error 160 0.13507 ** Significant at the 0.01 probability level, n.s. Nonsignificant at the 0.05 probability level. Table 17 ANOVA for light effect on starch content in mung bean leaf blades (Figure 7). Source df MS F Light (L) 2 0.10842 1.27 n.s Time (T) 4 0.99205 11.58 * LT 8 0.03668 0.43 n.s Error 142 0.08568 * Significant at the 0.05 probability level, n.s. Nonsignificant at the 0.05 probability level. 57 Table 18 The Effect of exogenous sucrose on mung bean leaf abscission. Light Treatments Sugar Treatment Red Far-red (Break strength. % of initial)^ Vacuum Infiltrated Control 103.2 X 29.7 y 0.3% sucrose 115.2 X 25.0 y 3.0% sucrose 95.3 X 24.9 y Sugar Solution^ Control 110.5 X 40.8 y 0.3% sucrose 93.8 X 25.5 y ANOVA Source df MS F Sugar (S) 3 370.0 2.3 n.s. Light (L) 1 33451.0 208.3 * * LS 3 161.9 1.0 n.s. Error 16 160.6 a Means of the same line followed by the same latter are nonsignificant at the 0.05 probability level by DNMRT. b Cuttings were infiltrated with sucrose solution at concentration as indicated. Control cuttings were infiltrated with distilled water. All treated cuttings were maintained in distilled water throughout the light treatments. c Cuttings, without vacuum infiltration, were placed into distilled water (control) or in sucrose solution during the light treatment. ** Significant at the 0.01 probability level, n.s. Nonsignificant at the 0.05 probability level. 58 CHAPTER 5 DISCUSSION 5.1 The Role of Auxin in the Light Control of Abscission Results of these experiments are consistent with previous reports (Craker ^ , 1987; Decoteau and Craker, 1983; 1984; Curtis, 1978) on the role of light in abscission. Treatment of leaf tissue with red light inhibited abscission, and treatment of leaf tissue with far-red light promoted abscission as compared with dark controls. In addition, the data support the concept that light control of leaf abscission is achieved through a phytochrome-mediated regulation of the internal level of a translocatable abscission inhibitor supplied by the leaf and active at the abscission zone. The differences in loss of break strength under dark and far-red light conditions between cuttings having a relatively large leaf blade area and a small leaf blade area are consistent with either a greater residual guantity of this inhibitor in the larger leaf or a longer lasting capacity for producing such an inhibitor in the larger leaf. Ethylene has been considered the most effective stimulator of abscission (Jackson and Osborne, 1970; 59 Osborne, 1989). Production of the gaseous plant growth regulator in mung bean cuttings was observed to be higher in far-red light treatment (Decoteau and Craker, 1987). Abscission enhancement by far-red light may be explained through the increased production of this hormone. Studies on the interactions of aminoethoxyvinylglycine (AVG, an ethylene synthesis inhibitor) or Ag+ (ethylene action inhibitor) with light quality indicated no relationship between ethylene production and the decrease of break strength of abscission zones, or between ethylene present and abscission (Decoteau and Craker,1987). Exogenous ethylene at physiological concentrations did not eliminate the inhibitory effect of red light on abscission (Decoteau and Craker, 1987). These results indicated that ethylene was not related directly to light control of abscission and another plant hormone may be involved. Auxin is a known inhibitor of abscission (Rubinstein and Leopold, 1964) . The differences between the possible photoinduced inhibitory effect of auxin and other plant growth regulators on abscission were separated by applica¬ tion of TIBA, an inhibitor of auxin transport, to the tissue. Application of TIBA reduced the retarding effect of red light on abscission and enhanced the dark and far- red light effect in causing abscission. These results 60 indicated that the abscission inhibitor produced in red light-treated leaves responds to the transport inhibitor as auxin does and that uninterrupted transport of the abscission inhibitor is essential for light control of abscission. Previous investigations have shown the effectiveness of exogenously applied auxin on retarding abscission in blade-free petioles and in other organs. Elimination of the red light effect on abscission by TIBA indicates that an auxin-like hormone was produced in the leaf tissue and translocated to the abscission zone. Further support for the induction of an endogenous abscission inhibitor by red light is evident for an examination of abscission regulating properties of diffusate collected from leaves in light and dark. The patterns of abscission in leafless petioles in dark and treated with leaf diffusate collected under red or far-red light or dark condition were very similar to those of previous light exposure (Mao and Craker, 1990). Leaf diffusate collected from red light inhibited the loss of break strength in abscission zones relative to the effect of diffusate collected from dark-treated leaves. Diffusate collected from leaves treated with far-red light did not inhibit abscission compared with diffusate from dark- treated leaves. Bioassay by bean curvature of the auxin 61 activity in the diffusate from red light treated leaves indicated that the amount of auxin in the diffusate was equivalent to about 10~^ mol lAA dm~^. The results of the two bioassays and those of the exogenous application of lAA to leafless petioles predicted the internal lAA concentration in red light treated leaves to be in the — R — 4 — range of 10 to 5 x 10 mol dm , the concentrations reported effective in other physiological processes, such as pea stem elongation (Galston and Baker, 1951) . Quantification by GC-MS confirmed the results, as the amount of lAA detected in the diffusate from red light treated cuttings was 4 times that from far-red or dark treated cuttings. The data imply that continuous red light actively promotes the production or transport of the endogenous abscission inhibitor in the leaf. Maintenance of leaf tissue in the dark may exhaust the supply of auxin or prevent the movement of auxin to the abscission zone, with abscission occurring as the level of auxin falls. Far-red light either accelerates the loss or more completely prevents the translocation of abscission inhibitor to the abscission zone than darkness does. Previous work (Craker et ad., 1987; Curtis, 1978) has identified phytochrome as the acting light receptor for 62 controlling abscission. The phytochrome control of auxin supply to the abscission zone could be maintained through regulating the levels of either input or output pathways to the pool of auxin in the leaf blade. The sources of the lAA pool are synthesis and hydrolysis of lAA conjugates. The observation of radioactive ^^C-IAA after supplying the tissue with 2-^'^C-tryptophan indicates that mung bean leaf blades are capable of synthesizing this hormone from tryptophan. The higher rates of conversion of tryptophan to lAA noted in red light-treated tissue compared with dark or far-red light treated tissue is consistent with the concept that control of lAA synthesis from tryptophan is a probable mechanism by which phytochrome regulates auxin levels at the abscission zone. Losses from the lAA pool may be by oxidation, conjugation, or translocation. The fact that cuttings with relatively large leaf areas do not lose break strength in the abscission zones as guickly as those with smaller leaf areas suggests that light inhibition of abscission is not through control of translocation since regulation of auxin transport probably would not depend on leaf area. Recent results in this laboratory (Wang et aT. , in preparation) have indicated that light has no direct control over lAA transport. 63 lAA oxidase is a group of enzymes catalyzing the oxidative decarboxylation of lAA to produce indole-3- methanol (Sandberg et ad., 1987) . The same level of lAA oxidase activity observed in mung bean leaf blades under all light conditions questions the assumption of phytochrome control on lAA oxidase proposed by Sharma and Malik (1978). Pollen tube growth in Campsis grandiflora was enhanced by illumination of red or blue light, and the control mechanism was proposed to be through lowering lAA oxidase activity in the pollens, as noted in blue light, and so causing a higher level of lAA in these pollens (Sharma and Malik, 1978). The same mechanism was suggested for the red light enhancement of pollen tube growth. Blue and red light, however, have quite different receptors in plant tissue in photomorphogenesis. Our results do not support the theory of phytochrome control of lAA oxidase, suggesting that phytochrome has no effect on lAA oxidase, or that lAA oxidase in pollen systems and mung bean leaf blades are under different mechanism of control. The lAA oxidation rate, theoretically, depends upon the oxidase activity and the concentration of substrate, lAA, available. The absence of differences in lAA oxidase activity in different light conditions (Table 10) suggests that phytochrome has no effect on the enzyme. At the same 64 level of lAA oxidase activity, a slightly higher rate of decarboxylation in red light-treated leaf tissue (Table 11) suggests the possibility of a higher level of substrate present in the tissue. This concept agrees with the high rate of lAA synthesis in leaf tissue (Table 9) and higher lAA level detected in the leaf diffusate from red light (Table 8). The whole scheme of lAA metabolism in mung bean leaf blades, however, cannot be drawn since little has been done on the pathway of synthesis and hydrolysis of lAA conjugates. The data support the concept that leaf tissue exposed to red light continually synthesize lAA. This continuous supply of free auxin at the abscission zone maintains the tissue in the Stage I of abscission (the phase insensitive to ethylene). This conclusion is supported by the demonstration that red light reduces the sensitivity of the abscission zone to ethylene (Decoteau and Craker, 1987). Light control of auxin level in leaf tissue occurs at the level of synthesis, whereas degradation and transport of the hormone are not affected by red or far-red light or by darkness. Lower levels of lAA detected in leaf diffusate from dark and far-red light treated leaves is proposed to be due to less free lAA available in the leaf tissue. 65 5.2 The Role of Cellulase and Carbohydrate in Leaf Abscission: The final steps in abscission process occur at the abscission zone, where cell wall degradation results in the separation of the abscising organ from the parent plant. Cellulase has been proposed as the enzyme responsible for hydrolysis of the cell wall. Cellulase activity measured in abscission zone tissue of coleus with leaf area of 1 cm^ was related closely to the changes of break strength in the abscission zone in different light treatments. In the absence of leaf blade tissue, the light effect on cellulase activity was diminished; red light no longer had the ability to inhibit abscission and maintain a low level of cellulase activity. These results indicate that light effects on enzyme activity are not based on direct control of the cellulase activity in the abscission zone tissue and that leaf tissue is essential to the process. The distance between the leaf blade and abscission zone further requires a chemical signal capable of movement between the two tissues. Auxin, reported effective in inhibiting the appearance of cellulase in the leaf abscission zone in Phaseolus and in orange fruit abscission zones (Goren and Huberman, 1976), is proposed to be the chemical signal involved in the light control of leaf abscission in coleus. 66 (^UJ/ ^[un 25.0 ^U9]U03 3:^DjpAqoqjD3 9soon|6 loujLu) 56 ■H ■H IP PI X -p <4-1 -p CP 33 p x: -P T5 0) -P CP -P XJ 0) I-1 0) o o c cn 03 P U 03 C O 03 (/} o 0) C/) D CP 03 •H -p -p u o X! rH T! c 0) c C E a C tP 0) 03 C 0) 03 03 0) w If this is true, then light control of leaf abscission can be explained in terms of phytochrome regulation of lAA output from the leaf tissue, followed by translocation of the lAA to the abscission zone where the lAA level regulates the abscission process by controlling the hydrolytic enzyme activity. Light per se has no direct effect on the hydrolytic enzyme activity in the abscission zone. The decrease in carbohydrate content of leaf tissue in this study was consistent with translocation, respiratory consumption, and lack of photosynthesis in leaves in dark or in light below the photosynthetic compensation level. High levels of starch and low levels of soluble sugars in the leaf tissue before abscission had been noted previously by Egli ^ (1980) in soybean. The mechanism(s) for maintenance of low levels of soluble sugars in leaves in our study is unknown. The patterns of carbohydrate utilization in leaf tissue treated with red light, far-red light, or maintained in the dark, were similar, suggesting total carbohydrate level in leaf tissue is not a factor affecting light control of leaf abscission. The importance of relatively high levels of glucose 6- phosphate content in red light—treated leaves than in far- red light or dark-treated leaves is still unclear. Glucose 67 6-phosphate composes 63% of the total hexose phosphates (Atkinson, 1988), bridges the metabolism of saccharides, being involved in such activities as hexose conversion, glycolysis, glyconeogenesis, and starch and cellulose synthesis and catabolism. Glucose 6-phosphate is also the sugar form entering the pentose phosphate pathway. The larger quantity of glucose 6-phosphate in red light-treated leaf blade may suggest a more active metabolism in non¬ abscising tissue that will remain part of the functioning plant. A previous report by Dharvan and Malik (1979) indicated that glucose 6-phosphate dehydrogenase was under phytochrome control. Slightly higher levels of glucose 6- phosphate in red light treated leaves might keep the tissue in more active metabolic state, whereas dark or far-red light-treated tissue might be disrupted. The bases by which red light treatment maintained higher levels of glucose 6-phosphate are, however, unclear. If the effect of light on abscission were due to changes in the total sugar content in the abscising organ, loading the tissue with sucrose could be expected to alter the abscission process. The effect of sucrose on Phaseo- lus vulgaris leaf petiole abscission has been observed to vary with external sucrose concentration, leaf age, and light condition (Biggs, and Leopold, 1957). Low sucrose 68 concentrations promoted and high sucrose concentrations delayed abscission in the P_^ vulgaris explants kept in the dark, but not in those in white light. These results have been interpreted to mean that high levels of photoassimilate in tissue masked the effect of added sugar, even though the explants were bladeless. The addition of 0.3% or 3.0% sucrose to the leaf tissue in our light system, however, did not change the response of leaf abscission to red or far-red light, suggesting that total sugar content in tissue is probably not the controlling factor in the effect of light on leaf abscission (Mao and Craker, 1990). Our results indicate the need for an alternative explanation of the observed abscission of plant organs previously associated with light and photoassimilate levels (Heindl and Brun, 1983; Schou ^ ^., 1978). While the high rate of abscission noted for soybean flowers and pods on the lowest portion of the canopy has been explained as due to a reduction in photosynthesis, a more likely explanation appears to be a decrease in the red to far-red light ratio in the lower canopy. Holmes and Smith (1977) have previously reported that the ratio of red to far—red light within a canopy decreases with an increase in leaf area index. Since abscission has been demonstrated to be 69 under phytochrome control (Decoteau and Craker, 1987; Curtis, 1978), a decrease in the ratio of red to far-red light in a plant canopy may be the functional equivalent of a far-red light treatment which affects lAA supply to the abscission zone as discussed by Mao and Craker (1990) and induces abscission. From these results, we conclude that light control of leaf abscisison appears at the level of phytochrome control of lAA synthesis and lAA supply to the abscission zone, light has no direct control over cellulase activity in the abscission zone, and that total sugar content in the leaf blade tissue is not a limiting factor in the light control of leaf abscission. 70 LITERATURE CITED Abeles, F.B. and Rubinstein, B. 1964. Regulation and leaf abscission by auxin. Plant Physiol. 39: 963-969. Addicott, F.T. 1982. Abscission. University of California Press, Berkeley, California. Addicott, F.T. and Lyon, J.L. 1973. 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