This dissertation has been microfilmed exactly as received 69-22,139
HANSEN, Dale J., 1939- EXPERIMENTAL INDUCTION OF TROPIC RESPONSES IN TRIFOLIUM FRAGIFERUM L. STOLONS.
The Ohio State University, Ph.D., 1969 Agronomy
University Microfilms, Inc., Ann Arbor, Michigan EXPERIMENTAL INDUCTION OP TROPIC RESPONSES
IN TRIFOLIUM FRAOIFERUM L. STOLONS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
Dale J. Hansen, B.S., M.S.
******
The Ohio State University 1969
Approved by
Adviser Department of Agronomy ACKNOWLEDGMENTS
I wish to thank Dr. Leo E. Bendixen for the help he has given me as my adviser; especially for the guidance given concerning my research and dissertation. This work was made possible by the teaching and research associate positions granted to me by the faculties of the
Botany and Agronomy Departments. All assiBtanoe was sincerely appre ciated. I am indebted to my wife, Ruth, for the many hours spent in supporting my graduate studieB and in typing this dissertation.
Dr. Richard Fopham kindly provided assistance and materials for the
anatomical studies presented in this dissertation.
ii VITA
Sept. 3, 1939 • * • Born — Idaho Falls, Idaho
June, 1963...... B.S., University of Idaho Mosoow, Idaho
1963-1966 ...... Teaching Assistant, Department of Biological Sciences, University of Idaho, Moscow, Idaho
1966* ••••••• M.S., University of Idaho Moscow, Idaho
1966-1967 ..... Teaching Assistant, Department of Botany, The Ohio State University, Columbus, Ohio
1967-1969 ...... Research Associate, Department of Agronomy, The Ohio State University, ColumbuB, Ohio
FIELD OF STUDY
Plant Physiology
iii TABLE OF* CONTENTS
Page ACKNOWLEDGMENTS ii
VITA iii
LIST OP TABLES VI
LIST OP FIGURES vii
INTRODUCTION 1
Chapter
I. LITERATURE REVIEW 4
Tropisma— A Definition The Test Plant Effect of Light on TropismB Fhytoohrome Gravity Geoelectrio Effect Oxygen and Carbon Dioxide Requirements Exogenously Applied Growth-Regulating Chemicals Gibberellins Auxins Ethylene Cobalt Morphaotins
II. MATERIALS AND METHODS 28
General Growing Conditions Application of Exogenously Applied Materials Liquids Gases Light Studies Red— far-red Photoperiod Light intensity Assays of Endogenous Materials Ethylene Oibberellin-like substances Anatoraioal Studies
iv TABLE CP CONTENTS— Continued
Page III. RESULTS 37
Erogenously Applied Growth-Regulating Compounds General observations Gibberellic acid plus cobalt Oibberellic acid plus Morphaotin IT 3233 Ethrel and ethylene-producing substances DC MU Altering atmospherio components Light Studies Red— far-red Photoperiod Light intensity Assays of Endogenous Materials Ethylene Gibberellin-like substances Anatomy of Strawberry Clover Stems
IV. DISCUSSION 63
V. SUMMARY 78
APPENDIX 81
LITERATURE CITED 82
v LIST OF TABLES
TABLE PAGE
1. Some Aspects of Plant Growth and Development Which Are
Affected by Red and Far-Red L i g h t ...... 12
2. Concentrations of CheraicalB Used on Prostrate Stolons. . . 30
3. Shading Effect Produced by Various Layers of Cheese
C l o t h ...... 33
4. Release of Ethylene from Bending Stolons ...... 60
vi LIST OF FIGURES
FIGURE PAGE
1. The typioal growth habit of Trifolium fragiferuin L. f variety
Salina...... 2
2. A skeletal presentation of porphyrin, some common bile
pigments, and phytochrome...... 13
3. An early bending stage of a prostrate clover stem induced
by GA3...... 38
4. A later stage of curvature ...... 39
5. A typical 90° bend promoted by GA^ ...... 40
6. A tropic effect other than stem-tip curvature...... 41
7. The effect of GA^ (1 g/l) on stolons...... 43
8. Bending of clover BtolonB as a result of movement...... 44
9. Stem-tip elevations of stolonB treated with 1 g/l QA^ and
1 g/l GA^ plus cobalt chloride...... 45
10. A comparison of tropic curvatures following treatment with -2 50 ppm ethylene or 3 x 10 M Ethrel treatments...... 47
11. Tropic curvature following IAA (1 x 10 ^M) application . . . 49
12. Tropic curvatures of stolons in the absence of Og, CO2, or
treatments with 10 DCMU...... 50
13. The path of least resistance ...... 52 LIST OP FIGURES— Continued
FIGURE PAGE
14» The Lending of stolons treated with red or far-red light. . 53
15. Effect of photoperiod on erect type of strawberry clover. . 54
16. Effect of photoperiod on the prostrate type of strawberry
clover...... • ...... • 56
17. A comparison of erect and proBtrate-type strawberry clover
plants under 24-hour photoperiod ...... 57
18. Upward curvature of stolons placed in darkness...... 58
19* Measurements of gibberellin-like substances from dark and
light-grown stolons with the barley endosperm bioassay . 61
viii INTRODUCTION
Gravity, light, and various chemical substances cause plant organs to bend. Isolation of the receptor mechanism in tropic responses and the identification of biochemical Btep3 leading to differential growth provide many challenging problems.
Prostrate and erect stems of Trifolium fragiferum L. (straw berry clover), variety Salina (Pig. 1) were used in the present study
on tropisms. Prostrate strawberry clover stems become erect when kept
in dim light or darknesB, when kept in a nitrogen atmosphere, when
covered with water, or when treated with gibberellic acid (OA^)
(Bendixen, 1960). The present study was conducted to understand why
the above phenomena occur and to explain why Borne strawberry clover
stems are prostrate and others are erect. Three specific areas of
research looked promising and were investigated* chemical-, atmos
pheric-, and light-regulation of tropisms.
Strawberry clover stolons become erect when treated with GA-^
but curve upward only slightly following an indoleacetic acid (IAA)
application. It was desirable to know if tropic responses could be
induced or inhibited in these prostrate stems with other growth regu
lating ohemioals Buch as ethylene, kinetin, and Morphaotin. Ethylene
evolution from bending Btolons was measured by gas chromatography.
Gibberellin-like compounds were extracted from prostrate and bending
stems and compared using the barley endosperm bioassay. Clover stolons 2
“ .. - Ti^ i .*■• .. t. jv' *■ ^ V. * .rj £aua&m*a% ^ ^ i r . r - ^ W
ft/ ■'■•■ ■ ■ s.tff*r,-.v’V:.,jsiiu*>*"fc- +* '••jnip w/TLdMl
. iavaiUNWBL. ««l«V r maAtamwummmum^iA ■ m^avo»Min!nwtnu3t jffiraMBnmnsmw .. ’te^3XSjfte£A£lxa&'i£.^^
1
Fig. 1.— The typical growth habit of Trifoliuin fragiferum L., variety Salina. The prostrate plant on the left demonstrates the growth form commonly associated with T. fragiferum. The erect plant on the right is a single—gene recessive mutant of the prostrate form. covered with water or kept in a nitrogen atmosphere curved upward. It was suspected that a limited oxygen or carbon dioxide supply was the indirect cause of the curvature. Experiments were conducted in which the atmospheric composition was varied to investigate this hypothesis.
Light quantity, quality, and duration were Btudied to discover why strawberry clover stolons become erect when kept in darkness but are prostrate in bright light. A microscopic comparison of tissues and cell types of erect and prostrate strawberry clover stems was carried out. The first ten elongated internodes were examined in transection in an effort to determine why one type growB erect and the other grows prostrate. Sufficient information was obtained from the studies on chemical-, atmospheric-, and light-regulation of tropisms to postulate a mechanism of bending. I. LITERATURE REVIEW
Tropisms— A Definition
Plant movements have been of interest to scientists Bince the early 19th Century (Knight, 1806). The movements of higher plant organs (in-..which the direction of movement is not determined by the structure) have been categorized using the suffix "tropic" with a prefix denoting the source of the stimulus. ThUB, the adjectives phototropic, geotropic, hydrotropic, thigmotropic, chemotropic, magno-
tropio, etc. characterize the movements of plant organs. Further complicating the study of bending effects, workers have categorized
types of a given stimulus. Words such as diageotropic, orthogeotropic, piagiogeotropic, ageotropio, positively geotropic, and negatively
geotropic have been used.
A tropic response is the curvature developed by a plant organ
resulting from a varied environmental factor (Wilkins, 1966). Tropic
movements, unlike nastic movements, are not predetermined by the
structural characteristics of the plant part involved. Two prefixes
are often coupled with the word tropic. The first prefix describes the
direction of growth of the plant part while the second prefix describes
the cause. Thus, the term diageotropic, describes an organ growing
perpendicular to the force of gravity, while orthogeotropic describes
an organ growing parallel to the force of gravity. Plagiogeotropic
denotes growth in some plane not parallel or perpendicular to the 5 force of gravity. Ageotropic is applied to plant organB not affected by gravity, while positively geotropic and negatively geotropic desig nate whether the curvature is toward or away from the force of gravity.
The Test Plant
Recent work on the bending mechaniBm of plant organa has centered around a few plant species. Tropic studies of stoloniferous plantB have been primarily limited to a few tropical plantsi Panicum purpuraBcens, Alternathera fiooidea, EleuBine indica, Commelina cayan- neusis, Portulaca oleracea, Mimosa senBitiva, and Cynodon dactylon
(Langham, 1941 and Palmer, 1956); lazy corn, Zea mays (Nickerson, 1962)}
strawberries, Fragaria chiloenBis (Thompson and Guttridge, 1959)* and.
Btrawberry clover, Trifolium fragiferum (Bendixen, i960).
Strawberry clover, because of its tropic sensitivity, was of
special interest. The inferences drawn from the study of this plant
contributed greatly to physiological questions concerning tropic
curvatures. Morphological characteristics of strawberry clover have
been described but no mention has been made of its anatomical features
(Hollowell, 1939). The growth habit is commonly prostrate, but an
erect mutant of a single-gene was reported in the variety Salina
(Bendixen et al., i960). Though prostrate stems commonly had inter
nodes of 5-10 cm. in length, erect mutant stems had internodes of only
5-10 mm. It was not uncommon for the recessive erect type to revert to
the prostrate form. Work with strawberry clover showed that prostrate
stolons became erect when placed in shade or darkness, when covered
with water, or when placed in a nitrogen atmosphere. From these 6 studies in which the atmosphere was modified, it was inferred that an oxygen deficiency evoked the tropic curvature (Bendixen and Peterson,
1962b).
High concentrations of GA^ applied to stems and leaves of straw berry clover stolons caused the prostrate type to become erect
(Bendixen and Peterson, 1962a). Similar gibberellin effects were noted in other Trifolium species (Finn and Nielsen, 1959? Brink et al., 1960;
Ormrod and Williams, 1960; and Fletcher and Martin, 1962) but none were as clear-cut as those from strawberry clover. Only a slight tendancy toward ereotness was noted, however, when the stolons were treated with any concentration of IAA. Experiments (Bendixen, i960) with exogenously applied GA^ and IAA led to the hypothesis that an interrelationship existed between auxins and gibberellins in regulating tropic behavior of clover stolons. The erect plants were postulated to have a supra- optimal auxin level and an optimum gibberellin level, while the pros trate plants had an optimum auxin level and a suboptimal gibberellin level.
Although literature on tropisms of stoloniferous stems is not abundant, it is evident that several different chemical mechanisms control the direction of growth. For example, Bermuda grass (Cynodon daotylon) commonly has rhizomes, stolons, and erect stems on the same plant (Hitchcock, 1951). Lazy corn, a prostrate mutant of Zea mays, does not become erect when placed in darkness or when treated with GA^
(Nickerson, 1962). Most of the tropical stoloniferous grasses studied seemed to be affected by light and gibberellin application similar to strawberry clover (Langham, 1941? Palmer, 1956; and Montaldi, 1967)* 7
Strawberry (Frageria ohiloensis) stolons, on the other hand, were affected only slightly "by the ahove treatments (Thompson and Quttridge,
1959).
There were some similarities "between tropic curvatures of strawberry clover and other processes which occurred in plants, such as the straightening of the bean hypocotyl, flowering of long— and short- day plants, and the movements of mimosa leaflets. These similarities will be discussed later.
Effect of Light on Tropisms
The literature on phototropism is voluminous and several reviews and books are available on the history of this subject (Withrow,
1959; Ruhland and Bunning, 19^2; BriggB, 1963; and Wilkins, 1966).
There are four theories concerning tropiBms caused by light. The first and most familiar is that of Cholodnv-Went (Cholodny, 1926 and Went,
1928). They postulated that the tropic stimulus induces lateral tranB- location of auxin across a photosensitive region producing an observed auxin differential. In the second theory Galston and Hand (1949) suggested that a differential inactivation or destruction of auxin occurs in light. Galston (1950) also responsible for the third theory that light inactivates some enzyme or cofactor which limits synthesis of auxins in the apical region of a particular plant organ.
As a consequence, an auxin precursor would presumably accumulate on the light side and migrate by diffusion to the dark side where conver sion to auxin would occur. Blaauw (1914) proposed a theory that light impinging upon a plant cell in some way affeots its growth rate, either 8 by stimulating or suppressing it. ThuB, in a unilaterally illuminated organ, the existence of a light gradient— as a result of absorption within the organ— would produce a light-growth reaction gradient.
A mass of evidence has accumulated regarding the role of auxin in the phototropio response (Briggs, 1963). With both phototropic and geotropic-induced curvatures, the auxin concentration on the elongating side was about 60$, as compared to 40$ on the less rapidly elongating side (Briggs, 1963; Gillespie and Thimann, 1963; Goldsmith and Wilkins,
19641 and. Wilkins, 1966). After critical analysis of published infor mation, it was suggested that a lateral transport of auxin occurred in geotropically stimulated shoots (Wilkins, 1966).
Several plant organs which exhibited tropic curvatures, including strawberry clover, showed little differential movement of auxin or little curvature to exogenously applied auxin. Zinsmeister
(i960) investigated the phototropic behavior of flower and fruit stalks of Cyclamen UBing long photoperiods and high light intensity. Under these conditions, flower stalks curved positively while fruit stalks curved negatively. Exogenous auxin application did not reverse the direction of growth nor was there any difference in auxin content. A definite deorease in the water permeability of the flower stalk was found on the illuminated side. Zinsmeister suspected that curvature was a turgor phenomenon. Cynodon dactylon L. stolons exhibited no bending when treated with IAA (Montaldi, 1967).
Light appeared to affect both the direction and rate of the geotropic curvature of plant organs. The curvature of stamens of Hosta oaerulea changed from positive to negative after exposure to light 9
(Pilet, 1950)* RhizomeB of Aegopodium podograria wore diageotropic in darkness, "but were positively geotropic for a short time after exposure to thirty seconds of red light (Bennet-Clark and Ball, 1951)* Vanilla rootB, diageotropic in darkness and in light (at wave lengths of between 5 5 0 - 7 4 0 nm), became positively geotropic after exposure to blue light (Irvine and Preyre, 1961). Seedlings of Sinapis alba grew more nearly vertical after exposure to red light than when kept in darkness
(Mohr and Pichler, 1961)* Vincetoxicum officinale was normally erect, but sometimes became twining when grown in the shade (Bequeudre et al.,
1966). Twining shoots were also obtained with experimental treatments of weak light, darkness, or gibberellic acid.
The influence of light in altering growth rates has been recog nized. Stem elongation in many species was inhibited during daylight hours (Sachs, 1872). In other species greater growth rates occurred during the day (Darrow, 1929| Thut and Loomis, 1944). The inhibitory effects of light on the rate of geotropio curvature of Avena coleoptiles
(Czapek, 1895 and Krones, 19H) and Phyoomyces aporangiophores (Czapek,
1895$ Pilet, 1956j and Dennison, 1964) were noted. Geotropic curvature of Avena coleoptiles waB decreased for sixty minutes following a fifteen-minute exposure to white light (Franck, 1951)•
The nature of the photoreceptive pigment in tropic curvatures is still largely unknown. Although it seems an easy task to ascertain the wave lengths of light essential for tropic stimulation to isolate the corresponding photosensitive pigments, this has not been the case.
Much of the very recent evidence supports the role of phytochrome in the tropic process, but evidence exists for chlorophyll, carotenoid, and flavenoid pigmentB as the light receptor (Briggs, 1963). Light- induced movement of the ohloroplast of Mesotaenium caldariorum was due mainly to light absorbed in the spectral regions of chlorophyll a and b.
Qalston and Baker (1949) have suggested that flavenoid pigments may be important in tropic curvatures, and recent work affirms this (Bara and
Oalston, 1968). Riboflavin is thought to be the photoreceptor in the oxidation of IAA. The absorption spectrum of riboflavin and the action spectrum for light activation of IAA oxidase showed cloBe similarity.
Ascorbic acid and dehydroascorbic acid were photooxidized using flavin mononuoleotide (FMN) as a photosensitizer in an aerobic atmosphere with catalase and ethanol to remove hydrogen peroxide (Haberman and Gaffron,
1957)* Flavins have also been implicated in the contact coiling of pea tendrils. High concentrations of quercetin-triglucosyl-p-coumarate were found in the tip of an uncurled tendril (Jaffe and Gal3ton, 1967).
During coiling a decrease of thirty per cent in the concentration of this flavenoid was observed. The active photoreceptor for the curva ture in Avena coleoptiles was thought to be of a carotenoid nature
(Shropshire and Withrow, i960). It is pointed out, however, that an attenuation of light across the eoleoptile by means of a secondary inactive pigment may complicate the process.
Phytochrome
The description of the red-light effectB on plants has been almost universally considered as a description of phytochrome actions
(Leopold, 1964), Plant responses to red light (660 nm) or far-red
light (730 nm) such as anthocyanin formation, unhooking of the hypocotyl 11 in emerging seedlings, light stimulation of leaf enlargement, and seed dormancy describe the phytochrome involvement in plant regulation.
Photoperiodic light effects are alBo thought to be mediated through this receptor (Hillman, 1967).
Early work concerning effects of light on plants was done on the daily length of the light period, a phenomenon called photoperiodism.
During the past few years it haB been realized that these early experi ments did little more than control the amount of red and far-red light which the plant received. Although it is not possible to ascertain whether the work on photoperiodism and the action of red and far-red light on plants is synonymous, it is apparent that the two effects are closely related.
The effects of red and far-red light were discovered by Flint and McAlister (1937)- They found that red light in the area of 660 nm caused lettuce seed germination while far-red light (710-750 nm) inhibited germination. Many plant responses have been discovered which are partially or completely controlled: by the action of red and far-red light. Lower forms of plantB, as well as vascular plantB, are affected by red and far-red light* A partial list of the effects of red and far-red light on angiosperms has been compiled (Table 1). Though most of these effects have little connection with tropic curvatures there is often a great degree of similarity between faotors which regulate the various types of effects.
Several major differences have been demonstrated between the effect of red light on the geotropio ourvatures of Avena (Blaauw, 1961) and Zea (Goldsmith and Wilkins, 1964)* With Avena, red light induced 12
TABLE 1
SOME ASPECTS OP PLANT GROWTH AND DEVELOPMENT WHICH ARE AFFECTED BY RED AND FAR-RED LIGHT
Wave Length of Light Causing Effect Investigator Effect
^Flowering in ahort day plants Garner & Allard, 1920 far-red Flowering in long day plants Garner & Allard, 1920 red Epiootyl elongation Downs, 1955 red Hypocotyl elongation Downs, 1955 far—red Internode elongation Downs, 1955 far—red Petiole elongation Downs, 1955 red Mesocotyl elongation Loercher, ^^66 far—red Straightening of pluraular hook Downs, 1955 red Bending of Avena coleoptiles Blaauw, 1961 red Bending of Zea coleoptileB Goldsmith & Wilkins, 196/ far-red Cotyledon enlargement Mohr, 1959 red Movement of root tipB away from a negatively charged surfaoe Jaffe, 1968 far-red Set of potato tubers Garner & Allard, 1923 far—red Onion bulb formation Magruder & Allard, 1937 red Increase in leaf aize Liverman & Johnson, 1955 red Seed germination Flint TABLE 1— Continued. Rave Length of Light Causing Effect Investigator Effect Increases in carotenoids of tissue cultures Godnev et al., 1967 red Lycopine pigment formation in tomato Piringer & Heinee, 1954 far-red Anthocyanin formation Siegelman & Hendricks, 1957 red Increase of ascorbic acid Schopfer, 1967 red Increases of phenylanine ammonialyase Scherf & Zenk, 1967 red Increased fat degradation in mustard cotyledons Karow & Mohr, 1967 red Increased virus content of Reinert & Kasperbauer, tobacco callus tissue 1966 red Production of nitrogen-fixing root nodules on legumes Lie, 1964 red *A11 work "before 1937 was on photoperiodic effects. Most of these experiments have been repeated to implicate phytochrome. 14 stem bending soon after treatment and the effect disappeared before twenty-two hours. There was some dispute as to whether this curvature in Avena was reversible with far—red light (Wilkins, 19^5)* In contrast red light inhibited the geotropic curvature in Sea, but the inhibition oocurred late in the bending process. Par-red light did counterbalance the effects of red light in Zea coleoptiles. There is little evidence to explain these differences between coleoptiles. The series of changes in geotropic responsiveness could reflect induced changes in the gravi- perception mechanism, in the growth rate of the tissue, in the effici ency and rate of the lateral transport of auxin or other substances, or in the phytochrome molecule. Many of the aspects of plant growth and development are con trolled by a reversible photochromic pigment which exists in two formst P , which has an action maximum near 660 nm, and P„ with an action r ir maximum near 730 nm. Absorption of light by either form converts it to the other form* p ______red light p r far-red light fr This photochromic pigment, phytochrome, is a biliprotein which is readily soluble under alkaline conditions and bears as a chromophore one or more bilitrine moieties that are closely related to the chromo— phores of the algal pigments phycooyanin and allophycocyanin. The structure of the phytochrome chromophore is closely related to other common bilitrine molecules (Pig. 2). Phycooyanin, a pigment similar to the phytochrome chromophore, crystallizes readily, is free of metals, and is in the stage of reduction between bilirubin and mesobilirubinogen 15 C - ^ N ^ “ C r - C )-n Porphyrin skeleton N N r e -crN Bile pigment skeleton M V M P P M M V Bilivirdin (green) 1OH H o DN = C •N' ‘N M V M P P M ME Phycooyanin (hlue green) ^ c J ^ L J-C - fsj -^\V|'0 n _ c ^.c-N-protein %-J M C M P P M M E Phytochrome (blue green) ^ - C ^ J=C ^ J- C= L ^ ml /protei n © +» N ■ffl i v-a c TJ a s 0) tH KCOOH C-R fn II >J< M *T M P P M M E Phytochrome (blue) - o U “C_”^ N ' ^ Pig. 2,— A skeletal presentation of porphyrin, some common bile pigments, and phytochrome. (M = methyl; E = ethyl; P - propyl; V » vinyl) 16 (White et a l ., 1964). By manipulating phycooyanin, a tentative struc ture has been worked out for phytochrome (Crespi et a l ., 1968). The molecular weight of phytochrome extracted from oat seedlings is about 60,000, but it is likely that different plants may have different phytochromes. Molecular weights aB high as 150,000 have been reported (Hillman and Purves, 1966). It is also possible that different phyto chromes exist in the same plant (Spruit, 1967)* Extraction of phytochrorae proved most satisfactory from dark- grown seedlings, and partial purification of extracts containing phyto chrome was quite easily achieved (Siegelman and Butler, 19^5)• A pure solution is difficult to achieve and at present is not possible in green plants (Mumford and Jenner, 1966). Before the discovery of phytochrome, it was predicted that the far-red absorbing form of the photomorphogenic pigment could revert to the red-absorbing form in the dark by an enzymatic prooesB. This proved to be true but the reversion was not complete and even when seedlings were grown in complete darkness, both the and Pfr forms of the pigment were detected (Linschitz et al., 1966). The dark reversion phenomenon of phytochrome is a very complete process with different conditions existing in raonocots than in dioots. According to Hillman (1967) "the process was insensitive to oxygen levels and metal-enzyme inhibitors, and in vivo kinetics and properties of dark reversion were quite different from those observed in extracts. The conversion of Pfr was demonstrated in Zea tissue when oxygen levels were reduced below lOjS (Butler and Lane, 1965). Pf conversion was also effected with carbon monoxide. Both oxygen and carbon monoxide conversion of Pfr 17 were approximately inversely proportional to the decrease in respiration so that at low respiration rates little was detected. The mechanism of phytoohrome action in tropic and other responses is not clear at the present time. There is some support for the theory that phytoohrome causes gene activationt setting in motion new synthesis of KNA and thenoe protein, which in turn results in the observed response (Yfeidner et a l . , 1965)* An alternative hypothesis, that the primary reaction of phyto chrome may be one of controlling membrane permeability, iB strongly suggested by work done with Mimosa (Pondeville et al.t 1966). Mimosa leaflets folded within five minuteB after an application of red light. Jaffa and Qalston (1968) recently worked out a proposed system for plant responses through membrane permeability which could be triggered by light effects, physioal stimuli, plant hormones, etc. A stimulus is sensed by an unknown receptor and ATPase is activated at the membrane. This results either in release of H+ ions from the cell, accompanied by any available anions, or the release of anions, accompanied by a cation, preferentially H • This release of osraotically active solute results in a loss of water from the adaxial cells into the adjacent vascular bundle. This leads to a loss of turgor in these cells and a contrac tion of the adaxial surface. Some consideration was given to the hypothesis that one or more formB of the phytoohrome molecule may act as an enzyme. Although little work has been done on this theory, it provides a possibility for explaining phytoohrome action. 18 Gravity The crux of the problem in geotropic curvature, as with other tropic curvatures, centers around the meohaniBm controlling the direc tion of curvature of the plant organ. Audus (1962) indicated that gravity influences plant organs or cellB only by virtue of the masbob of one or more of their component parts. Since an organ responds to gravity when supported along its entire length, geotropic growth curva tures cannot be the result of differential stresses set up on itB upper and lower surfaces arising from the cantilever effect of its own weight. Haberlandt (1900) and Nemec (1900) independently proposed that the falling of the heavier components of the cell cytoplasm, Buch as starch grains or calcium oxylate crystals, triggered the bending. Since 1900 many workers studying geotropisms have attempted to prove or disprove this Btatolith theory, and a summary of their findings is available (Brauner, 1954? Audus, 1962; and Wilkins, 1966). ¥hile the statolith theory is the best available at the present time to explain the effect of gravity on plants, its validity remains to be conclusively demonstrated. For example, Griffiths and Audus (1964), using electron microscopy techniques, demonstrated that amylo- plasts fall fast enough to cause bending of Vicia faba root tips. On the other hand, in root tips of Lepidium sativum, ten to twelve minutes were required for the starch grains to fall approximately half way across a cell (iversen et al., 1968). Movement beyond this point was negligible. These authors concluded that the erratic movement of the amyloplasts lends little weight to the starch statolith hypothesis. Geoeleotric Effect The development of a potential difference between the upper and lower aides of a horizontal plant organ waB first reported by Bose (1907) and has sinoe been described in more detail (Brauner, 1927* Clark, 1937? Schrank, 1947? Jantsch, 1959? and Grahm and Hertz, 1962). The lower side of an organ becomes positively charged with respect to the upper and a potential difference of up to 60 mV can be detected. The exact cause of this potential difference is unknown but the result of such a charge could bring about the migration of ionic materials within the cell. This fact is particularly useful in explaining the migration of the anion of the weakly ionized IAA molecule from the top to the lower half of a bending organ. More recent evidence, however, haB indicated that the transverse electric potential which develops after tropic stimulation is a result rather than a causal factor in the development of auxin asymmetry (Grahm, 19^4 a^d Wilkins and Woodcock, 1965). Oxygen and Carbon Dioxide Requirements The oxygen and CC>2 requirements for tropic curvatures have not been completely worked out. Carbon dioxide concentrations above caused a cessation of all growth and tropisms induced by gravity (Chapman et al., 1924). The water plant, Kymphaea, grew to the surface of the water due to a lack of C02 (Oessner, 1959). Strawberry clover stolons bent upward in a nitrogen atmosphere if light was supplied (Bendixen and Peterson, 1962b). When 02 was withheld from c o m coleop— tiles, but not completely evacuated, a moderate tropic curvature was 20 observed (Naqvi et al., 1965)» and coleoptile sections exhibited the usual tropic curvature (Dedolph et al., 19^5)« With the evacuation of air, however, neither corn nor oat coleoptiles curved geotropioally in anaerobic conditions (Wilkins and Shaw, 1967)* The directneBs-of-aotion of C02 and 02 on tropic curvature is unknown. For example, 02 deficiencies may produce the observed result through an alteration of auxin transport. An 02 level in the atmosphere of 1$ inhibited auxin movement by and in anaerobic conditions the movement was stopped completely (Goldsmith, 1968). The decrease in the activity of phytochrome as a reBult of decreased 02 levels (Hillman, 1967) may regulate the tropic curvature, and the role of C>2 in respira tion exertb a regulatory influence. The role of C02 in photosynthesis and its regulatory role in opening Btomata are well understood. Carbon dioxide may exert an indirect action on tropic curvatures through one or more of these pro cesses. A more direct correlation, however, exists between CC>2 and tropic curvatures. Increased levelB of C02 stimulated the opening of the bean hypo- cotyl hook (Kang et al., 1967). In the absence of C02 bean seedlings remained hooked, even in the presence of red light. Ethylene kept the bean hypocotyl hooked and it was postulated that C02 and ethylene were competitive inhibitors. ThiB competitive inhibition between C02 and ethylene has been confirmed in the effects on other plants such as abscission (Burg and Burg, 19^7? Burg, 1968). 21 Exogenously Applied Growth-Regulating Chemicals Gibberellins Tropic curvatures due to an exogenous application of gibber-*- ellins are limited and the first brief mention of possible tropic curva tures by gibberellins was made by Stowe and Yamaki (195?) and Stodola (1958)» should be pointed out, however, that almost all tropic research has been done with GA^ and many effects may have been missed* Bryophyllum crenatum tubers treated in autumn with GA^ developed long, orthotropic shoots, while control plants remained dormant or dev-* eloped slowly growing stolons with new tubers at the end (Dostal, 1959)* When one-node outtings of Bryophyllum crenatum were made, plagiotropic stolons developed. Exogenous application of QA^ induced the developing stems to grow erect. GA^ altered the growth habit of Trifolium pretense (Brink et al., i960 and Stoddart, i960). The stems of straw berry clover assumed a prostrate habit but could be induced to grow erect with GA^ application (Bendixen and Peterson, 1962a). Stolon differentiation, as well as the growth habit, of Fragaria chiloenBis could be regulated by GA^ (Thompson and Guttridge, 1959)* Tropic curvatures to GA^ have also been noted in Trifolium repens (Finn and Nielsen, 1959? Fletcher and Martin, 1962), Circoes intermedia (Dostal, 1959)t Oryza sativa (Maeda, i960) and Cynodon daotylon (Montaldi, 196?)- The effect of gibberellins on tropisms was thought to be nothing more than a stimulation of auxin production. However, GA^ caused the unhooking of the bean hypocotyl while auxins were inhibitory (Klein, 1965). Gibberellic acid, at a concentration of one part per 22 million, significantly increased the amount of the geotropically-induced curvature of Avena coleoptiles (Norris and Brotzman, 1965)* Indole- acetic acid increased curvature “both in light and darkness. Nhile most tropic effects resulted in an upward curvature following GA^ application, the reverse has been noted. A soil applica tion of GA^ to dwarf alfalfa caused the stems to become prostrate (Pauli and Sorensen, 1961). It is also of interest to note that not all prostrate plants became ereot when treated with OA^. Stem elongation and abnormal flower development of lazy corn occurred as a result of GA^ stimulation, but no upward bending waB noted (Nickerson, 1962). Auxins The first-known, naturally occurring growth regulator, indole- acetic aoid (lAA), which has a regulatory influence on tropisms has been well publicized. Though the action of this compound within the plant iB only slightly understood, much has been published concerning synthesis, degradation, and translooation as well as the regulatory role in physiological processes. No attempt will be made to review this extensive literature, and the pertinent information concerning this study has been previously mentioned. A much less publicized fact about auxins is that they do not induce tropio curvatures in all cases. Although the Avena curvature test has been a standard test for auxins, strawberry clover stolons (Bendixen, 1960), bean hypoootyl (Klein, 19^5)» and Cynodon dactylon (Montaldi, 1967) did not curve following auxin application. Ethylene Exogenous applications of ethylene cause defoliation of oertain plant species (Neljubow, 1901). Much is known concerning the effects of ethylene in the fruit ripening process. Since the advent of the gas chromatograph, it haB "been established that ethylene iB an endogenous regulator not only of fruit ripening (Burg and Burg, 1962), hut also of vegetative processes (Burg and Burg, 1966; Burg and Dijkman, 1967). The effect of ethylene on tropic curvatureB, in addition to epinastic curvatures, has been noted (doeschl and Pratt, 1965? Matsuraura and Hayashi, 1966). Ethylene produced in the hook region of etiolated pea plants kept the hook from opening (Ooeschl et al., 1967) and yet not enough ethylene reached the subapical zone a few millimeters away to cause any perceptible swelling. Ethylene caused an intense inhibi tion of growth on the lower side of a root (pisum) during the geotropic curvature, but only a light inhibition on the upper side (Chadwick and Burg, 1967). Much work has been done concerning the relationship between ethylene and auxin metabolism. An altered rate in the production and movement of auxin may account for at least part of the tropic curvatures. When ethylene was applied to intact plants the amount of diffusible auxin which could be recovered was considerably reduced (Guttenberg and Steinraetz, 1947? Valdovinos et al.. 1967). There were several possible explanations for this effect. 1. Ethylene may enhance auxin destruction* “While the auxin content of pea and Avena seedlings is not significantly lowered following ethylene application (Burg and Burg, 1966; Burg and Burg, 1967), the IAA oxidase activity of cotton is increased (Hall and Morgan, 1964). 2. Ethylene may inhibit auxin synthesis* Valdovinos et al. (1967) have indicated that the conversion of labeled tryptophan to IAA in light- grown pea and Coleus plants is greatly reduced if the plants have been treated with 25 ppm ethylene for 18 hours. 3. Ethylene may inhibit polar auxin transport* Although etnylene does not appreciably alter auxin uptake or transport under in vitro conditions (Abeles, 1966), it often inhibits dn vivo transport (Burg and Burg, 1967; Burg, 1968). Burg (1968) has summarized the many reports which indioated that increased amounts of ethylene were released from tissues treated with auxin. The biochemical pathway by which ethylene is produced is still unknown, as well as the cite of synthesis within the cell. It is known, however, that the addition of methionine to plant tissue incre ases ethylene production (Lieberman et al.. 1965? fang* 1967). Young tissues which were high in auxin content were likewise high in ethylene production (Burg, 1968). Ethylene caused growing cells in most roots and many stems to immediately reduce their rate of elongation and to expand instead in a radial direction (Burg and Burg, 1966; Chadwick and Burg, 1967)* Prom circumstantial evidence of this nature, one could hypothesize a role for 25 ethylene in the cell wall or in membrane permeability. Even though thie is an attractive theory for explaining the differential changes in cell size within a tissue exhibiting a tropic curvature, more evidence is needed before this conclusion can be reached. The discovery of 2-chloroethylphosphonic acid and its subsequent appearance under the name "Ethrel" (Amchem, 1963) has and will facili tate the Btudy of the effect of ethylene on plant tissues. The degra dation of thiB compound, marketed as a colorless, water-soluble liquid, has been worked out as followsi 0 0 Cl-C-C—P-OHU 2NaOHTci—C—C—P-CTNa+ r H ] 2H 4- _ H„C=CH„ + NaCl l ----- » I _ . * z z OH 0 Na + NaHPO-j + 2H20 This pH-sensitive degradation is probably not enzyme dependent since it occurs in the absence of catalysts as the pH iB raised (Varner and Leopold, 1969). Physiological responses to 2-chloroethylphosphonic acid are similar to those of ethylene. Cobalt Although cobalt has not conclusively been demonstrated to be an essential element for the vascular plantB, it does exert a regulatory influence upon their growth and development, including tropiBmB. Cobaltous ions caused the unhooking of the bean hypocotyl (Klein, 1959) and were implicated in IAA effects and phytochrome—controlled effects. Cobaltous chloride decreased the destruction of IAA in Pisum stems while manganese chloride increased IAA destruction (Galston and Siegel, 1954). Clarkson and Hillman (1967) reported that phytochrome synthesis in Pisum was inhibited by cobaltous nitrate. This fact had been 26 previously supported “by the findings of Salisbury (1959) that flowering in Xanthium, a short-day plant, was delayed by the application of cobalt. MorphactinB In i960 some interesting effects of fluorene-9-carboxylic acids on plants were observed at the biological research laboratories of E. Merck AG, Darmstadt, Germany. In 1964 these fluorene derivatives, which lastingly alter plant morphology, were called Morphactins (Schneider, 1964). COOH General Morphactin Structure While the effects of Morphactins included the loss of lateral bud dormancy, retardation of growth, and retardation of chlorophyll destruction, their effect on tropic curvatures was of particular signi ficance (Morphaotin Data-Sheet, 1965). Both the phototropic and geo- tropic curvatures of stems and roots were affected. Existing tissues became somewhat bent and distorted while developing tissues lost all sensitivity to the tropic cause. Thus, Khan (1967) found that seeds germinating in the presence of Morphactins developed in the plane in which the seed was placed. Morphactins are not naturally occurring metabolic components and their connection with the tropic mechanism is not well understood. Two of these Morphactin compounds have been given numbers, IT 3233 (n-butyl-9-hydrox3rfluorene-(9)-carfcoxylate) and IT 3456 (methyl-2-chloro- 9-hydroxyfluorene-(9)-carboxylate). They are in oommon use today. A decrease in the IAA-oxidase level in roots and an increase in shoots was observed following Morphactin application. An independence of action with GA^ has also been reported (Mann et al., 1966} Tognoni, 1967). The action of these fluorene derivatives was more pronounced on dicots than on monocots and they were relatively ineffective on corn (Tognoni et al., 1967). II. MATERIALS AND METHODS General Proving Conditions Single erect and prostrate olones of Trifolium fragiferum L.t variety Salina, were propagated vegetatively in growth ohambers. The growing conditions were maintained at 50$ relative humidity, a day temperature of 78°F| a night temperature of 72°F, and a photoperiod of * 12 hours. The light was provided by 22 cool white fluorescent tubes and six 60 watt incandescent bulbs which produced a combined intensity of approximately 3000 foot candleB at the level of plant growth. The early experiments of this study were conducted with plants grown in sand culture, irrigated at regular intervals (6 times per 24 hours) with half-strength Hoagland's solution. In later experiments the plants were placed in Bix-inch pots in a mixture of sand-soil-peat (1*1|1) supplied with nutrients as needed to maintain luxurient vegeta tive growth. One-half inch mesh wire was used as. a horizontal support for elongating stolons. In order to control the two-spctted spider mite, Tetranyohus telarius L., which periodically infected the chambers, Outhion, 0,0-dimethyl S-[4-oxy-1,2,3-benzotriazin 3(4H)-ylmethyl] phos- phordithioate, was used. In all experiments care was taken in handling and moving the Btolons since moving or tipping produced some tropic response. After Btolons were moved, a period of 48 hourB was allowed to elapse before work was continued. It was not possible, however, to follow this 28 procedure in all of the light studies. Application of Exogenously Applied Materials Liquids Aqueous solutions of desired composition and concentration were atomized onto the plant until no more liquid would adhere to the plant surface. Tween 20, at a concentration of 0.1^, was used as a wetting agent. Gibberellic acid, cobalt chloride, Morphactin IT 3233t kinetin, acetic acid, ethyl alcohol, Ethrel, DCMU (3-(3»4-dichlorophenyl)-1,1- dimethylurea), acetone, IAA, BNP (2,4-dinitrophenol), and methionine were atomized onto the plant in this manner. A list of concentrations used and the solvents needed in addition to water are listed (Table 2). From each series of concentrations, the concentration exhibiting the maximum tropic effect was then replicated in a randomized block design using at least five samples per treatment in each of four replications. All spray treatments were made at 2:00 P.M. since the length of the preceding photoperiod was critical. Visual observations of each treat ment and measurements of stem-tip elevation from the horizontal were recorded over a 72-hour period. Qases Prostrate plants were placed under eighteen-liter bell jars and subjected to air containing ethylene, air deficient in COor air deficient in Og- Chemically-pure ethylene was introduced into the bell jar by means of a gas-tight syringe to produce concentrations from 0.1 ppm to 100 ppm of ethylene within the chamber. The COg was removed from the air by passing a stream of air through a series of 4» 1-liter 30 TABLE 2 CONCENTRATIONS OP CHEMICALS USED ON PROSTRATE STOLONS Substance Quantity Substance Quantity -2 CoCl2 + GA3(lg/l) DCMU 10”? M 10-3 M 1 0 *"/i M 10 ' M * ^0~Z M * 10~c M 10 X 10 5 M Morphactin + QA, Acetone 10~^ M * 1-1 M (IT 3233) (Ig/l) 10 7 M M 10-2 1 0 ^ M 10 ^ M 10"5 M M io:a 10 4 M Kinetin 10"p N -2 10”‘ M IAA 10 i M 10 4 M 10“J M * 1 0 M • " d M 10 M 10 5 M -2 Acetic acid M DNP M -1 10 t 10 M io-* M 10 M M !-3 i°:| 10 M 10 5 M 10-4 M -1 Methionine M 10-2 Ethyl alcohol M 10 ? M -1 10-2 M M 10 M i o i M * -3 10 M 10“5 M -4 10 M Ethrel 3 i 10~2 M ' 3 i lo'i M * 3 x 10“^ M 3 x 10 M ♦Maximum tropic effect 31 Erlenmeyer flasks containing 500 ml 1 N NaOH. Barium chloride was used to check the effectiveness of the scrubber system. To provide an oxygen-free atmosphere, N2 and C02 from compressed gas cylinders were mixed, combining 0.03$ C02 with the N 2. Measurements of N2 and C02 ratios were made by a titrimetric method as the gaseB passed through 4 small water-containing vessels. In studies from which 02 or C02 were excluded, the flow rate was maintained at 10 to 12 liters per hour. Visual observations of each treatment and measurements of stem-tip eleva tion were recorded at various intervals during a 72-hour period. Light Studies Red— far-red The apparatus and procedures used for red and far—red illumina tion of plants have been described (Withrow, 1959 u^d Klein, 1964)* Prostrate strawberry clover plants were placed in an air-cooled, light tight chamber and covered with either a red (#650) or far-red (#750) plastic filter (purchased from Carolina Biological Supply Company). Each plant was irradiated for 3 minutes at the end of the regular 12- hour light period and then placed in darkness. Each filter, in addition to the desired band of monochromatic light, transmitted infrared radia tions. These undesirable infrared radiations were removed by covering the red filter with 10 cm of a 1$ OuSO^ solution and the far-red filter with 10 cm of distilled water. Five 150-watt reflector flood lamps were adjusted in distance from each filter to give a light intensity of 1.25 x 10 ergs cm sec beneath the far-red filter and 5 x 10 ergs — 2 — 1 cm sec beneath the red filter. Light intensity was measured with a 32 radiometer, YSI Model 6 5 (manufactured by Yellow Springs Instrument Company), The plants were left in darkness for 14 hourB then stem-tip elevation was recorded. Five plants were subjected to each of the red and far-red treatments and 5 plants were moved directly from the lighted chamber to darkness to serve as controls. The experiment was repeated 4 times. Photoperiod Twenty-four vigorously growing erect clover plants and an equal number of prostrate clover plants were out back to 1 cm from the soil and then subjected to either a 12 or 24-hour photoperiod. Light inten sity during the 12-hour photoperiod was 3000 foot candles followed by 12 hours of darkness. The light quality has been described under the sectioh "General Growing Conditions". Light intensity in the 24-hour photoperiod was 3000 foot candles for 12 hours as previously described, and the intensity during the following 12 hours was 100 foot candleB from six 60—watt incandescent bulbs. At the end of 3 weeks, observa tions of morphology and tropic curvatures were recorded and the experi ment was repeated. Light intensity Two experiments were conducted to ascertain the effect of light intensity on tropic curvatures of strawberry clover BtolonB. In one experiment an axillary bud near the base of a prostrate plant was allowed to develop while the other axillary buds were excised. This resulted in a plant with two stolons attached to the same root system. 33 One of the two etolonB was subjected to darkness by carefully inserting it through a small hole into a box covered with aluminum foil. The other stem was left exposed to the light regime of the growth chamber. After 72 hours, the stem-tip elevation of the two stolons was recorded. This experiment was repeated 12 times. In the other experiment, pros trate clover plants under 12-hour photoperiods were shaded with from 1 to 10 layerB of cheese cloth to ascertain the effect of light inten sity on the tropic prooess. Light intensity at the plant level after passing through the shading material is given (Table 3). TABLE 3 SHADING EFFECT PRODUCED BY VARIOUS LAYERS OF CHEESE CLOTH No. of Layers of Light Intensity at Plant Cheese Cloth Level (foot candles) 0 3000 1 1400 2 1000 3 800 4 670 5 580 6 510 7 440 8 390 9 350 10 320 Ten plants were subjected to each treatment and the stem-tip elevation was recorded every 24 hours for 5 dayB. Eaoh experiment was started at 2*00 P.M. and the most important treatments (from 4 to 8 layerB of cheese cloth) were repeated 4 times. 34 Assays of Endogenous Materials Ethylene To assay for ethylene production an 8 cm length of a prostrate clover stem was inserted through a notched slit in a No. 5 No. 6 rubber stopper. Any free space around the stolon passing through the slit was carefully filled with cotton. Aluminum foil was used to coat the inside of the rubber stopper which was then inserted tightly into the neck of a 125 ml Erlenmeyer flask. The stopper had previously been fitted with a half-hole rubber septum to facilitate the removal of an air sample for gas chromatographic analysis. At the end of 10 hours, a 10 ml air sample was removed from each flask with a gas-tight syringe and analyzed for ethylene with a Barber Coleman gas chromatograph. Ethylene evolution was measured from plants placed in darkness, from untreated control plants, and from prostrate plants treated with p ^ 1 g/l GA^, 3 z 10” M Ethrel, 10 M IAA. The results wore recorded as —9 X x 10 1 ethylene released per gram of fresh weight tissue per hour. The procedure for ethylene analysis has been described (Burg and Burg, 1962). The temperature of the 80-120 mesh alumina oxide column was maintained at 130°C after pretreating the column for 8 hours at 200°C. The temperature of the hydrogen flame detector was 150°C to prevent water condensation from the air sample. The flow rate of the carrier gas, helium, waB 50 ml per minute. Pure samples of ethylene and COg were used to oheck the authenticity of the unknown peaks and for running the standard concentration curves. 35 Qibberellin-like substances PetioleB and leaves were removed from light-grown prostrate stems and "bending stems kept in darkness for 48 hours. The youngest 10 cm section of each stem was excised, cut into small pieces, quick frozen with dry ice, and lyophilized. The tissue was then Btored in a desiccator until the gi"b"berellin-like substances were extracted. A modified extraction and separation procedure developed by Jackdon (1967) was used. A Virtis "23M homogenizer was used to grind 200 mg of the lyophilized tissue with 12 ml of methyl alcohol. During the 3-minute griiiding process a 4 ml aliquot of methyl alcohol was used to wash down the sides of the container. The ground sample was shaken vigorously for 30 minutes and filtered through Whatman #1 filter paper. The filtrate was combined with 12 ml of chloroform and 10 ml of water and centrifuged for 10 minutes at 2000 times the force of gravity. This procedure separated the colored pigments into the chloroform layer while the water-methanol layer contained the gibberellin-like materials. The water-methanol layer was decanted off and flash- evaporated to a volume I o b s than 1 ml. ThiB Bmall sample was spotted on a 1-inch wide Whatman #1 paper chromatogram. The chromatogram was developed in a descending manner with isopropyl alcohol and water (4*1) until the solvent front had moved 18 cm. The chromatogram was dried and sectioned into 10 equal pieceR to be tested with the barley endo sperm bioassay for gibberellin—like substances. Himalayan barley (produced at the Aberdeen Experiment Station, Aberdeen, Idaho) was usdd in the testing procedure. The bioassay for gibberellins developed by Jones and Varner (1967) was followed in detail. The experiment was 36 repeated 5 times and the results were recorded as yig amylase released per 200 mg sample. Anatomical Studies Ten internodes, starting with the first elongated internode behind the stem apex, were cut from erect and prostrate strawberry olover stems and placed in FPA (5 ml formalin, 5 ml propionic acid, and 90 ml 50$ ethyl alcohol). The material was then dehydrated in an ethyl alcohol series and imbedded in paraffin (m.p. 53-56°C). Toluene was used as an intermediate to facilitate infiltration. Transverse sections 10 y. thick were stained with safranin 0 and fast green (Jensen, 1962). Ten stems were sampled in each cane and observations of cambial development, lignification, and collenchyma formation were noted. Longitudinal sections of bending stolons were prepared by the same method to study the cells and tissues in the bending area. III. RESULTS Exogenously Applied Growth-Regulating Compounds General observations The quantitative measurement recorded in the study of exogen ously applied growth-regulating aompounds was the upward curvature of prostrate stolons* The bending took place 2-4 cm behind the Btem tip, the exact distance being dependent on rapidity of Btolon elongation. Curvature did not always result in a 90° bend (Figs. 3» 4, and 5)I therefore, the simplest parameter to measure was stem-tip elevation. Other plant movements were observed but not measured, such as the bowing of the oenter portion of a proBtrate stolon (Fig. 6), the reduoed angle of the petiole with the stem, and the folding of the leaflets. Although most of the materials used in this work caused pros trate stems to become erect, a few growth regulators (kinetin and alanap), the solvents (water, acetone, ethyl alcohol, and acetic acid), the surfactant (Tween 20), and the insecticide (Guthion) did not induce the upward bending. Several important variables influenced the results observed after application of the growth regulatory compounds. The earlier in the day an experiment was started, the more rapid was the bending. The nitrogen level was also found to be critical. Excessive amounts of NH^NO^ hastened the bending even though Rhizobium bacteria were active 37 38 Fig. 3.— An early bending stage of a prostrate clover stem induced by GA^. Fig. 4.— A latei*0stage of curvature. Not always did a tropic curvature result in a 90 bend. When the promotive souroe of curvature was removed, bending ceased within a few hours and the stem tip began to elongate in the plane of stem orientation. 40 I O Fig. 5.— A -typical 90 bend, promoted "by GA^- 41 Fig. 6.— A tropic effeot other than stem-tip curvature. Ethrel treatments oft^n resulted in a curvature in the center of the stem as well as the~=stem tip, leaving only a small portion of the stolon touching the wire support. 42 in nitrogen fixation. For example, reaction time of the G A y induced tropic curvature could be shortened from a mean time of 14 hours to 3 hours by applying the GA^ at 8 1OO A.M. rather than at 2i00 P.M., and by adding excessive amounts of HH^NO^ instead of relying on nitrogen fixa tion in the root nodules as illustrated (Fig. 7). When potted prostrate clover plants were moved from one place to another an upward bending of the stem tips often occurred. The bending induced by moving the plant could be observed within 3 to 4 hours, but the stem tip had returned to the horizontal position by the end of 36 hours (Fig. 8). Twenty-four of the 48 plants tested did not bend upward. The maximum curvature noted was 5 mm and was observed 18 hours after the stimulation. Greater curvatures were observed when the stolons were inverted, rotated, severely shaken, or wounded. Experi mental results were therefore collected only from whole, undisturbed plants. Qibberellic acid plUB cobalt The combination of 1 d 10 C0CI2 with 1 g/l GA^ decreased the reaction time of the tropic curvature (Fig. 9)» The effect of cobalt, however, was dependent on the presence of nitrogen-fixing bacteria. When an excess of NH^NO^ was supplied the cobalt treatment had little effect. Cobalt in the absence of GA^ had no tropic effect. Qibberellic acid plus Morphactin IT 3233 The application of Morphactin IT 3233 did not cause bending of prostrate olover stolons, but when high concentrations (10 ^ or 10 ^M) were applied with 1 g/l GA^ the expected upward curvature of the stem 43 4 .. o G A_ + NITROGEN 3 .. < > Lii G A UJ CL h- LJ K to 12 24 36 48 60 HOURS Fige 7 .— The effect of QA, (1 g/l) on Btolons. The lower line is the stem elevation of stolons treated at 2*00 P.M. with nitrogen supplied "by nitrogen-fixing bacteria. The upper line ie the measure ment following treatment at 8:00 A.M. with NH^NO^ added. represents themean. represents linethesolid and curvature the maximum line represents dotted The o 2 STEM TIP ELEVATION 2 3 .. .. Fig. 8.— Bending of clover stolonB as a result ofmovement. asa result stolonB cloverof Bending 8.— Fig. 12 HOURS 24 36 48 60 44 o and 1 g/l QA^ plus oobalt chloride. oobalt 1plus g/lQA^ and STEM TIP ELEVATION 4 -- 4 3 2 -- -- Pig. 9*— Stem-tip elevations of stolons treated with 1 g/lQA^ treated with stolons ofelevations Stem-tip 9*— Pig. 12 46036 24 HOURS G A GA 48 45 46 tip was inhibited. Morphactins were the only compounds tested which inhibited this bending. Some distortion of plant parts occurred and stem elongation was decreased. The angle of the petioles with the main o o stem was reduced from 90 to 15 and the leaflets did not completely open. Although no upward bending of the stolons could be observed following Morphactin treatment, some twisting of the stolons was evident as well as some slight stem movements to one side or the other. Any treatment which caused a tropic effect also caused the stolons to be light green in color. The Morphactin treatments, however, caused the stolons to remain dark green or even increased the intensity of the green color. Ethrel and ethylene-producing substanoes Applications of Ethrel upward bending of prostrate Btolons within 2 hours after treatment. Not only was initia tion of the effect rapid, but stem curvature and elongation were also rapid. Thus, the stem tip had elongated about 35 mm in 36 hours. After the stem tip was 35 mm or more above the supporting surface the stolon began to roll sideways, allowing the stem to return to the support. The graph (Pig. 10) of the Ethrel response is therefore misleading as the stems still elongated in an upward direction. As previously reported (Bendixen and Peterson, 1962a), IAA caused only slight curvature. IAA was studied in relation to its poss ible effect on ethylene production, and measurements of tropisms indi cated that bending began 2 to 4 hours after IAA application. A peak height of about 1 cm was reached by 18 hours and by 48 hours the effeot with 50 ppm ethylene or 3 x 10 M Ethrel treatments. M Ethrel 10 3 x or ethylene 50 ppm with STEM TIP ELEVATION 4 3 2 Pig. 10.— A compariBon^gf tropic ourvatures following treatmentfollowing tropicourvatures compariBon^gf A 10.— Pig. 12 ETHYLENE 24 HOURS ETHREL 36 48 47 60 48 ■teas completely gone and the stem tips were again horizontal (Pig. 11). The concentration of IAA (1 x 10 ^M) needed to produce the tropic curvature caused the leaflets to fold down for several hours following application "but they returned to an open position. Methionine, a precursor of ethylene, was .-applied to prostrate stolons hut the result was a small how in the center of the stolon with no upward curvature of the stem tip. Plants enclosed in a hell jar containing 50 ppm ethylene followed a tropic pattern almost identical with the Ethrel treatment. DCMU Prostrate clover stolons treated with 1 x 10 DCMU "became light green and began to bend upward in about 36 hours. Stem-tip elevation increased as a funotion of time hut slowed as energy reserves within the plant began to decrease (Fig. 12). Altering atmospheric components Stolons subjected to an atmosphere deficient in COg bent simil arly to plants treated with DCMU. Stems began to bend upward in about 38 hours and curvature slowed as energy reserves within the plant began to decrease (Pig. 12). Stem-tip elevation rarely reached more than 20 mm. Subjecting the plants to an anaerobic atmosphere produced a tropic curvature which began 15 "to 16 hours after O2 was excluded. As stem-tip elevation reached approximately 25 mm, growth essentially ceased and little curvature occurred beyond 60 hours following initia tion of anaerobic conditions. STEM TIP ELEVATION 4 2 -- -- i. 1—Toi uvtr olwn A (110~^M) application. x IAA curvaturefollowing Tropic 11.— Pig. 12 436 24 HOURS 860 48 49 0, rtetet rt C^ XM. 2 trithtreatments or C02, ICT^M 3XJMU. STEM TIP ELEVATION 2 -- 2 3 -- Pig, 12.— Tropic curvatures of stolons in the absence of0o, in theabsence stolons of curvatures Tropic 12.— Pig, 12 HOURS 24 O - DCMU 36 48 - CO 60 50 51 Light Studies Tho prostrate stems followed the path of least resistance in bright light (Fig. 13).and could not be induced to grov sideways when placed in unilateral light. All curvatures were in a vertical direc tion and the stolons were affected by light quality, quantity, and duration. Red— far-red Measurements of stem-tip elevation were made 14 hours after the stolons had been given a red-, far-red-, or control-light treat ment. Far-red light was more effective than red light in causing stolons to curve upward (Fig. 14) • Measurements from the far-red light treatment were significantly different at the 1$ level from those of the red light and control treatments. Photoperiod The morphology of prostrate and erect strawberry clover plants kept under 12-hour photoperiods has been described (Bendixen, i960). The morphology of both types of clover, however, was drastically altered under a continuous photoperiod when 12 hours of the light were from incandescent bulbs with an intensity of 100 foot candles. The length of an average internode on the erect plant, having elongated for a 3—week period, vaB 3-5 cm long instead of the usual 5-10 mra (Fig. 15)* The oolor of the erect plants had ohanged from dark to light green. Under this continuous light system the prostrate plants were no longer prostrate. All stems had elongated in an upright direction and the 52 Fig. 13.— The path of least resistance. Stolons under a 12- hour photoperiod of 3000 foot candles gradually bend downward due to the weight of the stem. light. Stolons were given a 3-minute treatment of red or far-red or ofred treatment 3-minute a given were Stolons light. only white light followed by darkness. followed by light white received only plants Control 14for hours.in darkness placed and light STEM TIP ELEVATION M M IO-. 5 -- Pig. 14.— The bending of stolons treated with red or far-redor red with treated stolons of Thebending 14.— Pig. FAR - RED 1/ RED CONTROL 53 54 1 Fig. 15.— Effect of photoperiod on erect type of strawberry clover. The plant on the left reoeived a 12-hour photoperiod of 3000 foot candles of light for a 3-week period. The plant on the right received a 24—hour photoperiod (12 hours were 3000 foot candles fluorescent and incandescent light and an additional 12 hours were 100 foot oandleB incandescent light) for a 3-veek period. 55 internodeB were about the same length as those of the erect plants (Fig. 16). Stem diameter was decreased in these formerly prostrate plants and they assumed a lighter green hue. 'When the two types were compared after the 3-week continuous light treatment, the total height of the erect plants was slightly greater. This was due to longer petioles on the erect plants (Fig. 17)« In some instances the erect and prostrate plants could not be distinguished. When the two types of plants were kept under continuous fluor escent light with an intensity of 3000 foot candles, all stems devel oped shorter internodes with increased diameter. The erect plants remained erect and the prostrate plants remained prostrate except in a few instances where flowering was induoed. The prostrate type then exhibited a slight degree of erectness. Under these conditions the plants were all dark green. Lipfrt intensity The light intensity study was undertaken to observe the effect of light quantity on prostrate plants and to find the photosensitive plant organ. When prostrate clover plants were subjected to total darkness at 2*00 P.M., the initial bending occurred about 14 hours later (Fig. 18). Prostrate plants having two stolons attached to a common root system always remained prostrate when one stolon was kept in darkness and the other was exposed to the 12—hour photoperiod of the growth chamber. Plants kept in the growth chamber but shaded with cheese cloth failed to bend when the light intensity was above 600 foot candles. Fig* 16.— Effect of photoperiod on the prostrate type of strawberry clover* The plant on the right received a 12—hour photo- period of 3000 foot candles of light for a 3—week period* The plant on the left is the same clone but received a 24—hour photoperiod (12 hours were from 3000 foot candles fluorescent and incandescent light and an additional 12 hours were_lj)0 foot candles incandescent light). Both plants have grown for a 3-week period* 57 Pig. 17.— A comparison of erect and prostrate-type strawberry clover plants under 24—hour photoperiod. The plant on the left is from the erect-type clone and would be erect but have short internodes under a 12-hour photoperiod. The plant on the right is from the clone of the prostrate type and would be prostrate but with longer internodes under a 12-hour photoperiod. ° S STEM TIP ELEVATION 3 2 - -- Pig. 18.— Upward curvature of stolona placed in darkness.in stolonaplaced of curvature Upward 18.— Pig. 12 24 HOURS 36 48 60 58 59 When light intensity was less than 500 foot candles, the stolons Beamed to "bend at ahout the same rate whether the light intensity vas 0, 320, or 500 foot candles. The removal of leaves and petioles did not inhibit the tropic curvatures of Btolons placed in darkness. An aluminum foil cap over the stem tip, likewise had no effect in regula ting the tropic curvature in either light or darkness. Once a stolon was induced to hend with a grewth-regulating compound such as GA^ or ethylene, no regulatory influence of light could "be detected except as carbohydrates were provided through photosynthesis. Assays of Endogenous Materials Ethylene Ethylene evolution was measured from plants placed in darkness, from untreated control plants, and from prostrate plants treated with 1 g/l GA^, 3 x 10”^M Ethrel, 10~^M IAA. The results showed that a —Q — 1 — 1 small quantity of ethylene (2.4 x 10 1 hr g fresh weight) was released from control plants. Stolons treated with GA^ or kept in darkness did not differ-significantly from untreated plants in ethylene evolution. IAA, on the other hand, oaused an approximate 3~ _9 -1 fold increase in ethylene evolution, yielding 6.9x10 1 hr g fresh —9 —1 —1 weight. Enormous quantities of ethylene (430 x 10 1 hr g fresh weight) were evolved from plants treated with Ethrel (Table 4)« 60 TABLE 4 RELEASE OP ETHYLENE PROM BENDING STOLONS _9 _1 Treatment i 10 a hr g fresh weight GA, 2.4 Bark 2.1 Control 2.4 IAA 6.9 Ethrel 430.0 Qibberellin—like substances The gibberellin-like compounds extracted from prostrate clover stems kept in darkness or in light stimulated the release of <* amylase from barley endosperm. Host of the biological activity noted with the barley endosperm bioassay ocourred between an Rj. of T to 9 on the paper chromatograph (Pig. 19). A slight amount of activity was recorded at R^ 3, but this was primarily from plants kept in darkness. Control readings taken from barley endosperm tissue inoubated in the absence of any plant extracts ranged between 50.0-60.0 pg of » Recovery from the extraction prooedure was less than 70$. Anatomy of Strawberry Clover Stems Although the first elongated intemode behind the stem tip of both plantB (erect and prostrate clover stems) contained 13 to 14 rudimentary vascular bundles and were almost indistinguishable, the second and third internodes began to show some structural differences which were well defined in the fourth internode. These differences 61 3 0 0 -- cr" QH 2 00.. UJ > o -I o 1 I 00.. j' \ J U- ■TT'l > - o > > >' ? . Ir > IP > ■> > p?i CD > ‘ i >' ' ) ■ 5 y ■] >: „ j o k Bsj- ■ L * l !i Fi-j V Air. 300 UJ CO < 100 CD JfctAi i ikJ. 2 3 4 5 6 CHROMATOGRAPH FRACTION Fig. 19.— Measurements of gibberellin—like substances from dark and light-grown Btolons with the barley endosperm bioassay. (top portion light grown, bottom portion dark grown) 62 could be followed through to the tenth internode. Fasoicular cambial activity was oheerved in the lower part of the first elongated internode or in the early second internode of "both plant types. By the third internode interfascicular camhial activity waB evident in the prostrate stem type hut was absent in the erect one. Lignification of the interfascicular xylem cells soon followed in the prostrate type. Interfascicular activity was found in the erect type helow the fourth internode hut cell differentiation, including ligni- fication, was not found between the bundles even as far down as the tenth internode. The cambium within the bundles of the erect stem was very active and many layers of secondary cells were observed. Both stem types were hollow due to separation of the pith during growth. Collateral vascular bundles of the erect type contained a thick collenchyma bundle cap external to the phloem. Bundles of the prostrate type likewise contained a collenchyma bundle oap, but with fewer numbers of cellB and thinner cell walls. IV. DISCUSSION Few facts have been collected, to explain why plants produce stolonB, rhizomes, tuberB, or other plant stems which do not grow completely erect. While most of the theories concerning the direction of stem growth have been based on the unequal distribution of auxin or the displacement of large particles to the lower Bide of a cell, they are inadequate to explain the growth of plant organs parallel to the force of gravity. ThiB study was conducted to explain why strawberry clover stolons become erect when placed in darkness, when subjected to a Ng atmosphere, or when treated with high concentrations of GA^. It was hoped that a hypothesis could be formulated to explain how the direction of growth was regulated in prostrate stems. In general, geotropic or phototropic stimulation leads to an asymmetric delivery of endogenous auxin to a receptor placed at the base of excised organs (Briggs et al., 1957? Gillespie and Briggs, . 14 1961). A two to three-fold increase in the lateral movement of C — labeled IAA has been demonstrated in coleoptile sections (Goldsmith and Wilkins, 1964)* However, few measurements if any have ever been made on plant organs which are relatively insensitive to auxin appli cation. In addition, the lateral movement, if it occutb, may be of little consequence. Strawberry clover stolons fall into the category of plant organs responding only slightly to auxin application. 63 64 Plants release ethylene following auxin application (Burg, 1968). The slight tropic curvature elicited by IAA in strawberry clover stolons may have been due to the release of ethylene. Compari son of the stem-tip elevation curveB of ethylene, Ethrel, and IAA (Figs. 10 and 11) revealed that the reaction rates do agree, with only the extent of stem-tip elevation differing. This difference can be explained by the unequal amounts of ethylene present in the tissues (Table 4). TheBe results have been supported by the finding that ethylene prevented the straightening of the hypocotyl hook of pea and bean seedlings (Kang et al., 1967)* Auxin application was likewise inhibitory and ethylene release was detected following auxin applica tion. The present studies indicate that auxins play a limited role in tropisms of strawberry clover stolons and that their major effect is through ethylene release. A rapid and pronounced effect of ethylene on strawberry clover stolons was expected from previous tropism studies with ethylene. Ethylene produced in the hook region of etiolated pea plants caused the hook to close even though the concentration was insufficient to produce any perceptible swelling in the subapical zone a few milli meters away (Goeschl et al., 1967)* Similarly, ethylene caused an intense inhibition of growth on the lower Bide of pea roots during a geotropic curvature, but only a slight inhibition on the upper side (Chadwick and Burg, 1967)* It was suspected that ethylene would prevent any upward curvature in strawberry clover stolons. This hypothesis was supported by the knowledge that GA^ and ethylene commonly exert opposing actions (Scott and Leopold, 1967) ®A^ 65 very stimulatory to the tropic curvature. This hypothesis, however, proved to he wrong and ethylene caused handing even more rapidly than OA^. Although ethylene oommonly prevents stem elongation (Burg and Burg, 1966), in this instance stem elongation was greatly enhanced. The tissue, however, did heoome a lighter green color as is usually the case following ethylene application (Burg, 1968). The biochemistry of ethylene is not completely understood and from the physiaal properties of the gas, it is not possible to deter mine whether the biological action occurs in the lipid phase or else where in the cell. Because of the rapid upward curvature of the stolons, a logical hypothesis is that membrane permeability is differ entially affected. Reports of increased permeability have been made (Outtenberg, 1951? Abrams and Pratt, 19^7) but so have reports concerning the lack of increased permeability (Burg et al., 19^4| Lyons and Pratt, 1964). Though altered membrane permeability is a logical explanation for the action of ethylene upon the observed tropic curvature, there is insufficient evidence to support this view' and more work is needed. Evidence suggests that applied ethylene lowers the level of diffusible auxin by inhibiting auxin synthesis and transport and by enhancing auxin destruction (Burg, 1968). This does not, however, explain ethylene action in plant organs insensitive to auxin, such as strawberry clover stolons. Though ethylene caused bending of clover stolons it did not provide a common link between the various treatments evoking tropic curvatures. Insignificant amounts of ethylene were released when 66 plants were caused to bend by darkness or by GA^ application (Table 4)* There is some support for the idea that ethylene promotes nucleic acid and protein synthesis (Abeles and Holm, 1966). This leads to the syn thesis of degradative enzymes (Horton and Osborne, 1967)* vhioh in turn inoreaseB membrane permeability and the observed bending. After finding that phytochrome exerted an influence on the curvature of clover stolons, it was suggested that cobaltouB ions would prevent or inhibit the upward curvature of the stolonB. This belief wan sustained by the evidence that cobaltous ions caused enlarge ment of etiolated bean leaf disks in darkness (Miller, 1951)? inhibited phytochromes synthesis in Pisum (Clarkson and Hillman, 1967)» delayed flowering in Xanthium (Salisbury, 1959)» and caused straightening of the bean seedling hypocotyl (Kang et al., 1967). This hypothesis proved to be wrong and no inhibitory influence of aobalt on bending was observed. Although oobalt was ineffective in causing bending when applied alone, cobalt applied in conjunction with a GA-j treatment greatly enhanced curvature (Pig. 9)» Danilova and Demkina (1967) studied the effect of cobalt ions on the growth and development of legumes. They found that growth and development were affected but that the primary influence was through enhanced nitrogen fixation by root—nodule bacteria. The same effect may have been true in this study. Activity of the root—nodule bacteria vaB increased following an application of CoC^. "When excess NH^NQ^ was added with a GA^ treatment, a reaction rate similar to the cobalt—plus-0A^ treatment was observed (Pigs. 7 and 9). If cobalt inhibition occurred the effect was completely masked by the presence of Khizobium. To effectively 67 study the action of cobaltous ions on clover stolons it will he necessary to maintain Bterile conditions, at least vith regard to nitrogen-fixing organisms. The exclusion of C>2 and. COg from prostrate clover stolons caused the stolons to become erect (Fig. 12). A deficiency of 0^ evoked bending more than twice as fast as did the lack of CC^. Both curvatures are undoubtedly a result of secondary effects and an inter pretation of the mode of action is speculative. The exclusion of COg stimulated the upward curvature of prostrate stems at approximately the same time and rate as DCMU (Fig. 12). Since DCMU interrupts the transfer of electrons in photophosphorylation and has been widely used as a photosynthetic inhibitor (Levine, 1968), it iB believed that photosynthesis plays a crucial part in keeping the stems prostrate. ThiB belief is further substantiated by the fact that stolons ourve upward in darkness or in low light intensities. The photosynthetic action, however, does not have to be direct. This was demonstrated in the experiment in which connecting stolons, one in light and one in darkness, did not bend upward during a 72-hour period. Something was transmitted from the stolon in the light to the one in darkness to prevent bending. Possibly an abundant supply of adenosine triphos phate (ATP) or the reduced form of nicotinamide-adenine dinucleotide phosphate (NADP) is required to maintain reactions regulating the prostrate condition. Further experiments are needed to understand these relationships. Kang et al. (1967) found that CO2 promoted the straightening of the beam hypocotyl and that it opposed ethylene in this process. Carbon dioxide also induced a rapid decrease in the 68 permeability of HelianthuB roots to water. Within three minutes after COg treatment the influx and eflux of water from oells was depressed (Glinka and Reinhold, 1962). When the above two facts Eire compared from the standpoint of membrane permeability they appear to conflict. The results of this work with strawberry clover stolons are consistent with the findings of Glinka and Reinhold. As the COg level decreased, membrane permeability on the lower side of the stem could have increased and caused bending to occur. Many metabolic reactions in the plant are oxygen dependent and become altered as the Og supply is depleted. Although it has been demonstrated that tropisms do not occur in completely anaerobic condi tions (Wilkins and Shaw, 1967), tropisms do occur when a limited amount of Og is present (Dedolph et al., 1965)* Removal of Og or COg in the current experiments was not complete even though no Og or COg was supplied to the bell jar in the influent air supply. The chambers were not evacuated at the start of each experiment and some photo synthesis and respiration occurred. The conversion of the P^r form of phytochrome was demonstrated in Zea tissue when Og levels were reduced below 10^ or when carbon monoxide was applied. Both of these treatments inhibited respiration and Butler and Lane (1965) observed that the amount of P^r was approxi mately proportional to the rate of respiration. When the rate of respiration was low, little P^r waB detected. The active form of phytochrome involved in the bending of clover stolonB was Pr» The P^r form promoted the prostrate condition. Therefore, when Og was deficient the P^r form would disappear and the Pp form would increase. 69 Oxygen is also required to maintain a high rate of photosynthesis. This B t u d y demonstrated a role for photosynthesis in the prevention of bending. Few light studies have been conducted on prostrate plants. Langham (1941) concluded from his studies with light intensity that many prostrate tropical plantB are negatively phototropic in bright light. This statement was questioned by Palmer (1956) who repeated Langham's experiments and concluded that prostrate plants were not negatively phototropic in bright light. Bending as a result of unilateral bright light was not detected in the present work, but stolonB were only kept under such conditions for 48 hours. Plants grown entirely in bright unilateral light may have responded differ ently. All curvature occurred in one direction— up. In bright light the young prostrate stems in the seedling Btage would grow erect for some time and then gradually lean over. The stems did not appear to be negatively phototropic but rather ageotropic and aphototropic in bright light, growing along the path of least resistance. No treat ment caused the stolons to bend in a positively geotropic direction. The studies in which light quality was regulated, implicated phytochrome as a major photoreceptor. When stolons were subjected to a brief exposure of far-red light and then placed in darkness, bending occurred more rapidly than when stolons were given a similar treatment using red light (Fig. 14). 'Fh© red light treatment was not signifi cantly different from the control. Such a response would indicate that the Pr form of phytochrome plays a role in regulating the ereot- ness of stolons while P^r contributes to the prostrate condition. 70 Although red and far-red light studies contribute only circumstantial evidence, they are usually accepted aB a phytochrome response (Hillman, 1967). At present the separation of the phytochrome pigment from green tissue is extremely difficult due to the presence of other pigments and the very small concentration of phytochrome. Phytochrome extraction iB usually attempted only in dark-grown seedlings (Mumford and Jenner, 1966). Had the receptor pigment been extracted and a light absorption spectrum plotted, the above conclusion would bo more concrete. The photoperiodic studies of both erect and prostrate olover BteraB lend Bupport to the role of phytochrome in regulating the direc tion of growth, Erect and prostrate plants in bright light of either 12 or 24-hour photoperiods continued to grow erect or prostrate, respectively, as long as the ratio of red to far-red light was high. Under these conditions stems remained thickened with short internodes. In photoperiodB in which the ratio of far-red light waB increased by the use of incandescent bulbs, all stems grew erect and somewhat etiolated. When light intensity was reduced to a low level, or dark ness, there was an apparent shift in the ratio of P^ and P^r in the direction of Pr. Under these oonditionB prostrate stems became erect. Evidence exists for a regulatory mechanism of tropisms which is mediated by high intensity light. This system appears to be independ ent of the photosynthetic and phytochrome effect. Strawberry clover plants were prostrate when light intensity was high but became erect when light intensity fell below 580 foot candles. Attempts were futile in separating the effects of photosynthesis and phytochrome by adjusting 71 light intensity. When light intensity was below 58O foot candleB, bending occurred at about an equal rate even though light intensity was varied from 58O "to zero foot candles. Chemical reagents are often stored in dark-colored containers to prevent molecular rearrangement by light. It is, therefore, expected that similar compounds in living organisms would be rearranged in bright light. Living organisms possess dynamic metabolic Bystems capable of restoring these compounds to the original state or of producing new ones. Thus, an equilibrium would be established between several forms of the same molecule. Experimentally induced tropic curvatures were evident in prostrate clover stems at three separate timeBj 2-4 hourB, 14-16 hours, and 36—38 hours. Ethylene or materials releasing ethylene -caused bending which was initiated soon after the start of an experiment (2-4 hours). Removal of light or oxygen and treatment of plants with GA^ initiated bending in about 14—16 hours after the start of an experiment. Blockage of the photosynthetic process with DCMU or the absence of COg initiated bending in 36—38 hours* The short reaction times are more likely to be a direct change in membrane permeability followed by turgor pressure ohanges. The slower reaction rateB would allow suffic ient time for new protein synthesis and altered enzyme production. Information of this nature is often misleading and must be interpreted with care. The time differential may indicate only the speed at which a compound enters a plant stem and reaches the reaction site, or it may indioate the amount of time required to stop or start a given reaction occurring within the plant. For example, ethylene and OA^ may both directly alter the same system but ethylene would diffuse to 72 the site of action many times faBter than GA^ due to the disparity between physical properties. Or, on the other hand, both compounds may regulate the same system but the action of one substance may be direct and the other indirect. The observed result by either method would produce a disparity between reaction rates. Combining reaction rates from several treatments in an attempt to identify receptor mechanisms is probably futile. The anatomical studies were helpful in understanding differences between prostrate and erect strawberry clover stems at the tissue level. Transactions of both erect and prostrate stems from the first elongated internode to the tenth showed differences which grew pro gressively more striking. Though the first elongated internode of both stem-types was nearly indistinguishable in cross-sectional view and contained an approximately equal number of vascular bundles, caution must be used in making a direct comparison. Direct comparisons of lower internodes are even more questionable. A great disparity of internode lengths existed between the two types even though the tissue was about the same age. Also, stem elongation of the erect plant nearly ceases after 6 to 8 internodes have developed. Then, apical dominance is lost and the axillary buds near the base of the plant begin to grow. In the prostrate plant, apical dominance is incomplete from the early stages of development but the apical end of the main stem continues to elongate rapidly. Though both stem-types demonstrated interfascicular cambial activity below the third internode, no differentiation of the tissues in this area occurred in the erect stem. This was true even as far down as the tenth internode. In the tenth internode the vascular "bundles were large but separate. In contrast, cells in the interfasci cular region of the prostrate stem "became differentiated soon after they were produced. Lignin deposition in the xylem oells was especially evident. With the present knowledge it is difficult to explain the vascular development found in the erect stem. Differentiation of xylem oells in Coleus parenchyma tissue with only Blight lignification was demonstrated when GA^ levelB were high and IAA levels were low (Hansen, 1$66), The erect stem used in this study probably did not contain excessive amounts of gibberellin-like materials since GA^ applications stimulated great increases in internode length. The internodes were very short. The promotion of cambial acitvity, as well aB lignification, by IAA has been demonstrated (Fosket and Roberts, 1964). The intermediates of the biosynthetic pathway leading to the formation of lignin are known for the most part (Brown, 1969)* The factors which control lignification are poorly understood and appear to vary with environmental conditions (Phillips, 1954)t developmental stage (Stafford, 1965)1 and plant species (Stafford, 1967)* ThiB study and others (Cheng and Marsh, 1968) demonstrated that variations in lignification also ocour within specieB. The erect stems contained a large amount of collenchymatous tissue having heavily thickened walls. This tissue formed a cap external to the phloem of each vascular bundle. In the prostrate stems this collenohymatous tissue contained fewer oells and the cell walls were less thiokened. Duchaigne (1955)t described mature collenohymatous tissue as a strong flexible tissue consisting of long overlapping cells with thickened, nonlignified walls. Collenchyma cells, although having thickened primary cell.wallB, contain an active protoplast at maturity and are capable of further elongation and division (Esau, 19^5)* T*1© cell walls are rich in cellulose and pectic materials and contain much water. Collenchyma formation has received little attention and has been studied mostly from a mechanical standpoint. Collenchyma cell walls of Datura plants became thickened when the plants were exposed to wind (Walker, i9 6 0), He further observed that wall thickness was inversely proportional to the rate of stem elongation. The latter finding helps explain the large amount of collenohymatous tissue in short erect stems and the small amount in prostrate ones. The presence of gibberellin-like substances in Btrawberry clover has been previously demonstrated by means of the dwarf corn bioassay (Bendixen, 1960). The current study, using the barley endosperm bioassay, alBO demonstrated the presence of gibberellin-like substances in stems of strawberry clover. It had been hoped that a quantitative comparison of gibberellin levels between dark and light-grown clover stolons could be made. This was not possible due to insufficient recovery rates in the purification procedure. Since the barley endo sperm bioassay is relatively insensitive to gibberellin A^ (Jones and Varner, 1967) and since gibberellin is frequently found in plant extracts (West and Phinney, 1959? Macmillan et al., i9 6 0) the method has limited value. Better results oould be obtained if the barley endosperm bioassay were used in conjunction with another assay system. 75 Crozier and Audus (1968) were able to distinguish some definite qualitative differences in gibberellin content between light and dark- grown bean seedlings using the barley endosperm bioassay. Their findings are in harmony with those of this study but are much more pronounced. By using a phosphate buffered celite column to separate the various fractions of the gibberellin-like materials sharp peaks of biological activity were found in the eluate fractions number 3 and. number 7 * The dark-grown seedlings contained more biologically active materials than the light-grown seedlings in fraction 3 and the reverse vaB true in fraction 7- PerhapB the best method of studying gibber- ellins is by means of gas—liquid chromatography. Cavell et_al..(196 7) identified and quantitated 17 different gibberellins by this method. It appears that the various factors causing a tropic curvature are perceived by one or more receptors within the plant. Gravity is the most important of the factors regulating the direotion of straw berry clover stem growth. The other factors, such as photosynthetic effects, phytochrome effects, high-light intensity effects, and hor monal effects modify the gravitational effect. By comparing the inter action of the above factors a hypothesis can be fomulated for tropisms of clover stolons. An integral part of the hypothesis is that permea bility of various parts of the cell plasmalemma must be increased or decreased by gravity. This assumption is part of the statolith theory and the altered permeability is explained by the falling of heavy particles such as starch grains onto the cell membrane. The statolith theory is a limited approach to the effeot of gravity on plant cells. 76 For example, the entire weight of the cytoplasm restB on one surface of the membrane. The other surfaces are supported by turgor pressure. Furthermore, in an aqueous system, such as plant oells, nonpolar mole cules may separate to specific areas of the cell, depending on their density. A concentration of such materials would influenoe membrane permeability. Assuming that gravity favors increased permeability on the lower side of a cell, a group of cells in series would intensify the effect. Under these conditions a Btolon would bend upward as turgor pressure increased on the lower surface of the stem and decreased on the upper surface. If gravity were the only regulator of membrane permeability all strawberry clover stems would grow ereat. When gravity, acting upon the entire stem, caused the stem to bend sideways, the gravitational influence on membrane permeability would be altered and the fall arrested. Such an explanation may also explain the nutational phenomenon commonly associated with growing organs. Differential membrane permeability iB balanced by the effects previously discussed i.e. photosynthetic effects, phytochrome effects, high-light intensity effects, and hormonal effects. The stresses exerted in opposition to the gravitational effect can be less than or equal to the effect of gravity. When all forces are equal the stem grows as if the only force being exerted were the effect of gravity on the entire stem. When these forces are less than the effect of gravity stems grow erect. In the erect mutant these forces would always be less. The transduction of the various regulatory effeots opposing gravity into a common meaningful signal to regulate membrane permea bility is difficult to explain. The best explanation at present is 77 via the phytochrome molecule. Haupt (1968) haB provided evidence vhioh indicates that the P^r form of phytochrome found in membranes is oriented at right angles to the membrane while the Pr form is oriented parallel to the membrane. Perhaps not only light but chemicals can alter phytochrome forms creating different sized openings in the mem brane system. The future of research in this area looks promising. Although many important gaps still exist in our understanding of tropisms, progress is being realized. The complex nature of the tropic process is complicated by the modifying influence of the envir onment. Since a cell cannot be separated from its environment more research is needed to fully understand the tropic process. In undis turbed conditions plants grow in every direction and it is hoped that in the near future the biochemical and biophysical components regulating the direction of growth will be understood. V. SUMMARY Strawberry clover stolons curve upward when placed in dim light or darkness, when kept in a nitrogen atmosphere or covered with water, and when treated with high concentrations of GA^* Indoleacetic acid atomized onto the stems and leaves causes strawberry clover stolons to bend upward only slightly. This study was conducted to understand why the above treatments cause stolons to grow erect and to ascertain which biochemical reactions within the plant regulate the direction of stem growth. A single-recessive mutant of the prostrate form of Btrawberry clover is known which grows erect. Comparisons between the two stem types were made. Clover Btolone always bend upward and none of the treatments caused these prostrate stemB to curve in a negatively geotropic direction or to either side. The speed of the stem-tip curvature following GA^ treatment was dependent on the time of day the experiment was Btarted, whether the stolon had been moved, and on the amount of nitrogen available. Morphactin IT 3233 vas the only compound tested which completely prevented the upward curvature of stems following GA^ application. Ethylene and ethylene-releasing materials caused a rapid stem—tip curvature. The slight upward curvature of stolons to IAA was probably due to stimulated ethylene production. Stolons curved upward in the absence of Og or COg> Upward 78 79 curvature in the absence of Og was more than twice as fast as it was in the absence of COg. DCMU triggered the bending proceBB with about the same speed as low concentrations of COg. From the above results it can be inferred that photosynthesis partially regulated the prostrate growth habit of strawberry clover Btolons. Clover Btolons given a 3-minute far-red light treatment followed by darkness curved more rapidly than when given a corresponding treatment of red light or when placed directly in darkness. In a 12- hour photoperiod of incandescent and fluorescent light, both erect and prostrate plants continued to develop in the usual manner. In a 24- hour photoperiod (12 of which were from incandescent bulbs of a low intensity) all plants grew erect with elongated internodes. When the entire 24-hour photoperiod was of high intensity light the erect stems grew erect and the prostrate stems grew prostrate but with shortened intemodes and thicker stems. Whenever light intensity fell below 580 foot candles the prostrate stems became erect. It can be inferred that the form of phytochrome caused stolons to become erect. Light of high intensity caused stolons to assume a prostrate position. Ethylene evolution from bending stems was measured with the gas chromatograph. Insignificant quantities of ethylene were released from stems which curved as a result of darkness or OA^ treatments. Larger quantities of ethylene were released when bending waB evoked with Ethrel or IAA. The barley endosperm bioassay confirmed the presence of gibberellin-like materials in strawberry clover stems. The morphology of the erect and prostrate strawberry clover stems had previously been described in the literature, but the anatomy 80 had not been compared, A thick collenchyma bundle cap was found external to the phloem of each vascular bundle in the erect type. The collenchyma tissue external to the phloem of the prostrate type was composed of fewer cells and the oells had thinner walls than in the erect type. Both ereot and prostrate stems contained many secondary cellB as a result of cambial activity. The cells in the interfascicular area of the erect stem-type, however, did not differentiate into xylem or phloem cells. The more important factors influencing the direotion of Btolon growth are gravity, photosynthesis, phytochrome, high light intensity, and growth-regulating chemicals such as ethylene and GA^. Light and growth-regulating chemicals counterbalance the influence of gravi-ty. A logical hypothesis for the bending of stolons is that membrane permeability iB altered so that more solutes accumulate on the lower side of the stem causing greater elongation. The exact location of phytochrome within the cell needs to be determined aB well as the chemical changes which occur when it is irradiated with red and far-red light. The relationship of photosynthesis to the bending process and the energy retirements need to be determined. In order to prove the hypothesis concerning bending as a result of altered membrane permea bility, it will be necessary to find out where ethylene biosynthesis occurs within the cell and how ethylene reacts with cell membranes. Evidence to support the hypothesis can be obtained by measuring the movement of solutes into cellseon the lower side of the stem. The composition and structure of cellular membranes as they are .affected by gravity need further study. 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