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ISOLATION AND IDENTIFICATION OF A CYTOKININFROM MEDICAGO SATIVA L. (ALFALFA, ZEATIN, HPLC).
Item Type text; Thesis-Reproduction (electronic)
Authors Fimbres, Anna Maria.
Publisher The University of Arizona.
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Uni
Internationa! 300 N. Zeeb Road Ann Arbor, MI48106
1325567
Fimbres, Anna Maria
ISOLATION AND IDENTIFICATION OF A CVTOKININ FROM MEDICAGO SATIVA L.
The University of Arizona M.S. 1985
University Microfilms
International 300 N. Zeeb Road, Ann Arbor, Ml 48106
Copyright 1985
by
Fimbres, Anna Maria
All Rights Reserved
ISOLATION AND IDENTIFICATION OF A CYTOKININ
FROM Medicacro sativa L.
by
Anna Maria Fimbres
A Thesis Submitted to the Faculty of the
DEPARTMENT OF PLANT SCIENCES
In Partial Fulfillment of the Requirements For the Degree of
MASTER OF SCIENCE WITH A MAJOR IN AGRONOMY AND PLANT GENETICS
In the Graduate College
THE UNIVERSITY OF ARIZONA
19 8 5
Copyright 1985 Anna Maria Fimbres
STATEMENT BY AUTHOR
This thesis has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the copyright holder.
SIGNED ;
APPROVAL BY THESIS DIRECTOR
This thesis has been approved on the date shown below:
iU,,'J' A1^2/ ?. ns-5 R. G. McDaniel Date
Professor of Plant Sciences
ACKNOWLEDGMENTS
I am most grateful to Dr. R. G. McDaniel, Dr. P. G.
Bartels, and Dr. R. Matsuda for their supportr direction,
and encouragement. Thanks go to Dr. A. K. Dobrenz for
supplying the plant material used in this study. Thanks
also go to Dr. F. R. H. Katterman for his continued interest
in this project and for the many helpful suggestions.
I would like to thank my parents, Ray and Alice
Fimbresr for their support and unknowing influence. I am
also grateful to Patrick for his many suggestions and
solutions.
Finally, gratitude is expressed to all affiliates of
110 who have made this project a whole lot easier in many
ways.
iii
TABLE OF CONTENTS
LIST OF ILLUSTRATIONS vi
LIST OF TABLES viii
ABSTRACT ix
INTRODUCTION 1
LITERATURE REVIEW 4
History of Cytokinins 4 Chemistry of Cytokinins ....... 7 Biological Activities ........ 10 Responses to Stress 12 Extraction, Purification, and Identification 15
Extraction 15 Purification 17 Identification 17
Bioassays 17 High Temperature Effects on Alfalfa ... 20
MATERIALS AND METHODS 23
Plant Material 23 Sampling 23 Extraction Procedures 23 Testing of Extracts 27
Ultraviolet Spectroscopy .... 27 Thin Layer Chromatography ... 27 Bioassays 30
Barley Leaf Senescence . . 30 Radish Cotyledon .... 31
High Performance Liquid Chromatography 31
RESULTS AND DISCUSSION 34
Ultraviolet Absorption Spectra .... 34 Reverse Phase High Performance
Liquid Chromatography 51 Thin Layer Chromatography 68 Barley Leaf Senescence Bioassay .... 72 Radish Cotyledon Bioassay 72
iv
V
TABLE OF CONTENTS—Continued
SUMMARY AND CONCLUSIONS 75
REFERENCES 77
LIST OF ILLUSTRATIONS
Figure Page
1. Structures of some of the more common cytokinins ... 9
2. Ultraviolet spectrum for zeatin 36
3. Ultraviolet spectra for eluants of the Lew cultivar obtained by extraction procedure one 37.
4. Ultraviolet spectra for eluants of the Low Photo cultivar obtained by extraction procedure one 38
5. Ultraviolet spectra for eluants of the Lew cultivar obtained by extraction procedure two 40
6. Ultraviolet spectra for eluants of the Low Photo cultivar obtained by extraction procedure two 41
7. Ultraviolet'spectra for eluants of the Lew cultivar obtained by extraction procedure three 42
8. Ultraviolet spectra for eluants of the Low Photo cultivar obtained by extraction procedure three 43
9. Ultraviolet spectra for eluants of the Lew cultivar obtained by extraction procedure four 45
10. Ultraviolet spectra for eluants of the Low Photo cultivar obtained by extraction procedure four 46
11. Equation expressing cation exchange between functional group of column and a cytokinin 49
vi
vii
LIST OF ILLUSTRATIONS—Continued
Figure Page
12. HPLC chromatograms for eluants of the Lew and Low Photo cultivars obtained by extraction procedure one 56
13. HPLC chromatograms for eluants of the Lew cultivar obtained by extraction procedure two 58
14. HPLC chromatograms for eluants of the Low Photo cultivar obtained by extraction procedure two 59
15. HPLC chromatograms for eluants of the Lew and Low Photo cultivars obtained by extraction procedure three 60
16. HPLC chromatograms for eluants of the Lew cultivar obtained by extraction procedure four 62
17. HPLC chromatograms for eluants of the Low Photo cultivar obtained by extraction procedure four 63
18. Attempted standard curve for the barley leaf senescence bioassay, utilizing kinetin as the active compound ... 73
19. Attempted standard curve for the radish cotyledon bioassay, utilizing zeatin as the active compound 74
LIST OF TABLES
Table Page
1. A few of the sources from which zeatin and zeatin riboside have been isolated 6
2. A more comprehensive list of some of the sources in which cytokinins have been shown to occur . . 8
3. List of commonly used bioassays .... 19
4. Summary of extraction procedures used based on leaf tissue amount and the type of extraction medium used 28
5. List of sample eluants and their dilution factors 29
6. List of alfalfa leaf eluants subjected to HPLC 48
7a. Results of error analysis for area differences of HPLC peaks from day to day 53
7b. Results of error analysis for area differences of HPLC peaks due to spiking 53
8. Dilution factors for samples subjected to HPLC 55
9. Peak areas and Mg equivalents of zeatin for each sample eluant subjected to HPLC 66
10. Rf values for eluants subjected to TLC 69
viii
ABSTRACT
Extraction of zeatin from two cultivars of alfalfa,
Lew and Low Photorespiration, was attempted. Pour different
extraction procedures were employed in the isolation of
zeatin from alfalfa leaves. Identification of zeatin was
accomplished through the combined results obtained from
ultraviolet spectroscopy, high performance liquid
chromatography, and thin layer chromatography. Attempts at
determining the biological activity of isolated fractions
using the barley leaf senescence and the radish cotyledon
bioassays ended in failure. Thus the compounds isolated and
identified as zeatin in this study can at best be classified
as zeatin-like compounds until ascertainment of whether they
are biologically active or not has been accomplished.
Extraction procedure four was by far the most
successful in the recovery of zeatin-like compounds from
alfalfa leaves.
Significant differences between the two cultivars in
the amount of zeatin-like compounds extracted could not be
determined due to the variability inherent to each
extraction procedure. Suggestions for reducing this
variability to allow such differences to be measured are
discussed.
ix
INTRODUCTION
Effects of high temperature on cytokinin levels in
plants is a relatively underdeveloped topic of research.
These levels have been reported to decrease in response to
elevated temperatures (Skene and Kerridge 1967; Gur, Bravdo,
and Mizrahi, 1972; Zieslin, et al. 1984), but the exact
mechanism involved has not been disclosed, whatever the
mechanismr an important correlation between cytokinin levels
and heat resistance was formulated by Cole (1969). He
reported a maintainance of cytokinin levels by heat tolerant
pea seedlings as opposed to nontolerant pea seedlings in
response to heat stress. His data also supports the
suggested possibility that cytokinins are the important
chemical mediators which elicit the characteristics of high
temperature resistance in plants (Richmond and Lang 1957).
A plant whose cytokinin levels are maintained under high
temperature stress should also exhibit maintainance of
nucleic acid and protein metabolism (Chibnall 1939; Richmond
and Lang 1957).
"Summer slump" is a term used in the southwestern
United States which refers to decreased forage yield in
alfalfa during the summer months. This decrease in forage
yield has been linked to high temperatures (Robison and
Massengale 1967, 1968; Foutz 1973) and the metabolic effects
1
2
they elicit (Pulgar and Horton 1974; McDaniel, Dobrenz, and
Schonhorst 1981). The search for a way of sustaining alfalfa
productivity through this "summer slump" period has resulted
in little success. If one could clone a heat resistant
individual, then perhaps a population could be attained
whose productivity would remain constant throughout the
summer months. How would one select such an individual? An
obvious choice would be to select for individuals that are
capable of maintaining their cytokinin levels (and thus
their metabolic functions) under high temperatures.
It was originally suggested that screening for
individuals capable of maintaining their cytokinin levels
under heat stress could be accomplished by sampling
individuals grown in the field throughout the summer months,
and measuring the changes in their cytokinin levels by means
of bioassay techniques. This was quickly proven a fruitless
and unresourceful approach in view of the fact that little or
no characterization of the cytokinins present in alfalfa has
ever been attempted.
The focus of these experiments was to attempt the
isolation and identification of at least one cytokinin,
namely zeatin. This particular cytokinin was selected on
the basis of its reactive nature in several bioassays
(Letham 1967, 1971). Several extraction procedures are
employed for the isolation of zeatin from alfalfa leaves and
are compared. Identification of the cytokinin is achieved
3
by means of ultraviolet spectroscopy, high performance
liquid chromatography, and thin layer chromatography.
Biological activity of the isolated substances is
ascertained by means of the barley leaf senescence and the
radish cotyledon bioassays.
LITERATURE REVIEW
History Cytokinins
Cytokinin research began with Folke Skoog in 1941.
Using the technique of plant tissue culture, Skoog and Tsui
(1948) found that tobacco stem segments would not grow in
the absence of auxin, but that when 3-Indole acetic acid was
added cell enlargement and cell division occurred.
Jablonski and Skoog (1954) found that when vascular tissue
was removed from the tobacco stem segments, so that only the
pith was incubated in auxin treated medium, cell enlargement
took place without cell division. Extracts of the vascular
tissue could restore cell division. Attempts to isolate the
unknown substance from the vascular tissue gave inconsistant
results. Naturally the search was on for other substances
that could elicit cell division in the same way. Several
natural sources were probed and several promoted cell
division in the tobacco pith assay.
The first active compound to be isolated and
identified came from autoclaved herring sperm DNA. It was
structurally determined to be 6-furfurylaminopurine or
kinetin (Miller, et al. 1955, 1956). Since other compounds
endogenous to plants were found that showed the same kind of
activity as kinetin the general name kinin was adopted (Zwar
4
5
et al. 1963; Bottomley et al. 1963). To avoid confusion
with the kinins of mammalian biochemistry, the term
cytokinin (in accord with the effect on cytokinesis) was
proposed as a better alternative and has been used ever
since (Letham 1963; Skoog, Strong, and Miller 1965).
The discovery of kinetin sparked researchers to look
for cytokinins in plant tissues. It was not until 1963,
nine years after the discovery of kinetin, that the first
naturally occurring cytokinin was discovered by Letham. He
isolated zeatin from immature kernels of Zea mays (Letham
1963) and determined its structure (Letham, Shannon, and
MacDonald 1964, 1967). It took 60 kg of sweet corn kernels
to obtain 1.0 mg of zeatin in crystallized form. A more
detailed analysis by Letham (1973) detected the presence of
zeatin riboside and its 5%-monophosphate. Bioassay and
chromatographic methods have since been used to identify
zeatin and zeatin riboside in a range of plants. For
example, they have been identified in plum fruitlets (Letham
1966), in leaves and xylem of sunflower plants (Klambt
1968), sycamore sap (Purse, et al. 1976), and in coconut
milk (van Staden and Drewes 1975). Zeatin and its riboside
have also been found in fungi (Miller 1967) and bacteria
(Chapman, Morris, and Zaerr 1976). Other botanical sources
from which zeatin and its riboside have been isolated are
listed in table 1.
The second cytokinin isolated from natural sources
6
Table 1: A few of the sources from which zeatin and zeatin riboside have been isolated.
HORMONE SOURCE
zeatin Ztt. mays kernels
cotton ovules
tomato xylem sap
Cgrnybacterium
zeatin Zea mays kernels riboside
cotton ovules
chicory root
coconut liquid endosperm
tomato xylem sap
REFERENCE
Letham, 1966
Shindy and Smith, 1975
Van Staden and Menary, 1976
Scarbrough, et al., 1973 Armstrong, et al., 1976
Letham, 1968; 1973; 1974
Shindy and Smith, 1975
Bui Dang-Ha and Nitsch, 1970
Letham, 1968; 1974
Van Staden and Henary, 1976
7
was identified as N®-(2-isopentenyl)adenine. It was found
to occur as a base of t-RNA's in yeast (Biemann, et al.
1966). Other cytokinins isolated from non t-RNA plant
sources include dihydrozeatin, dihydrozeatin riboside,
glucosyldihydrozeatin, glucosylzeatin, and glucosylribosyl-
zeatin. Their occurrence in plants has been discussed in a
review by Horgan (1978). Table 2 contains a more
comprehensive list of sources in which cytokinins have been
shown to occur.
Chemistry &£ Cytokinins
All naturally occurring cytokinins known are N 6-
substituted adenine derivatives (see fig 1). More
specifically, it can be said that all naturally occurring
cytokinins are derivatives of isopentenyladenine, with one
exception, 6-(o-hydroxylbenzylamino)-9-B-D ribofuranosyl
purine. This compound was isolated from mature poplar
leaves (Horgan, et al. 1975). The effect that substituted
adenines have on cell division depends on where the site of
substitution is. N^-substituted adenines are known to
promote cell division, and it appears that an unsubstituted
1-position is a requirement for cytokinin activity (Skoog,
Hamzi, and Scweykowska 1967). It also appears that the
biological activity of cytokinin glycosides and glucosides
is weaker than those of the free purine bases.
The nature of the substituent attached to the 6-
Table 2: A more comprehensive list of some of the sources in which cytokinins have been shown to occur.
ORIGIN PLANT REFERENCE
endosperm corn Killer (1961), Letham et al. (1964) coconut Letham (1968)
fruitlets apple Zwar et al. (1963) peach Powell and Pratt (1964) plum Letham (1966)
seeds lettuce Barzilai and Mayer (1964) sunflower Miller and Witham (1964) pea Rogozinska et al. (1965)
v lupin Koshimizu et al. (1967) watermelon Maheshwari and Prakash (1967) pumpkin Gupta and Maheshwari (1970)
seedlings pea Zwar and Skoog (1963) leaves corn Miller and Witham (1964)
begonia Heide and Skoog (1967) cambium poplar Nitsch and Nitsch (1965) roots corn Miller and Witham (1964)
sunflower Weiss and Vaadia (1965) chicory Nitsch et al. (1966)
root exudate grape vine Loeffler and Van Overbeek (1964) sunflower Kende (1964, 1965) maple Nitsch and Nitsch (1965) cocklebur Carr and Burrows (1966) lupin Carr and Burrows (1966) pea Carr and Burrows (1966) balsam Carr and Burrows (1966)
callus tissue soybean Miura and Miller (1969) tobacco Wood et al. (1969)
crown gall periwinkle Wood (1964) prickly pear Wood et al. (1969)
9
J"! "Ne=e'°,,pH mt-GHf Ncitj
ft c m nCA u
•CO" -»J ' n/ H
B
H Vc""" -airtH**"*
r^Y^« 1 i y™ -s^v 1
|tib*«&
CM. ON fCt\\p(1
"SA*'0' « I
fttkosc.
E F
Figure 1: Structures of some of the more common cytokinins. A) kinetin B) zeatin C) zeatin riboside D) isopentenyladenine E) dihydrozeatin F) dihydrozeatin riboside
10
position of the purine ring directly determines cytokinin
activity. Highly active cytokinins have an exocyclic
nitrogen at the 6-position of the adenine ring, a
substituent size of five carbons, the presence of
unsaturation in the N6-substituent, a hydroxyl group at the
4-position of the isopentenyl side chain. It is these
chemical distinctions which have allowed zeatin to be
recognized as the most active naturally occurring cytokinin.
Both the cis and the trans isomers of zeatin occur
naturally? however, the cis isomer is found only to occur in
t-RNA, whereas the free cytokinin is found as the more
active trans isomer.
Biological Activities
What exactly does one mean by activity? Relative
activities of some of the chemical compounds discussed are
based upon numerous bioassay techniques, which will be
discussed later. In nature, however, cytokinins elicit a
wide range of responses some of which may be regarded as
"activities". The first response they elicit is an obvious
one: cytokinesis, the basis for their discovery. One must
keep in mind that their influence on cell division was a
discovery made using tissue cultures and there is little
direct evidence they elicit the same response in intact
plants. The fact that they are often present in high
concentrations in meristematic tissues would lead one to
11
believe that indeed they do play a role in regulating cell
division. There have been some results which support this
hypothesis (Letham 1963), and some which do not (Kende and
Sitton 1967). Cytokinins are also known to retard aging in
many plants. Many researchers have succeeded in delaying
senescence by the application of kinetin (Richmond and Lang
1957). Their exact mode of action in delaying senescence
has not been determined, but it is thought that cytokinins
directly influence protein and RNA metabolism or catabolism.
Based on their results, Richmond and Lang (1957) and Osborne
(1962), proposed that cytokinins retard senescence by
stimulating RNA and protein synthesis. Tavares and Kende
(1970) found just the opposite; that cytokinins delay
senescence by inhibiting protein and possibly RNA
catabolism. This last hypothesis is supported by data at
the level of free amino acids and enzymes in detached leaves
which have been aged with or without cytokinins. However,
their mode of action appears more complex than this
hypothesis would suggest.
For awhile it was thought that perhaps retardation
of senescence by cytokinins was a result of the
translocation of assimilates to the cytokinin treated area.
Mothes, et al. (1959) discovered that localized treatment of
detached tobacco leaves with kinetin resulted in the
attraction of metabolites from the untreated part of the
leaves. Kinetin, however, retards senescence in isolated
12
leaf discs as well as discs connected to an untreated disc
(Osborne 1962). Thus, there does not seem to be a
correlation between the translocation of assimilates to a
kinetin treated area and the delay of senescence in that
treated area.
Cytokinins also elicit some control of
morphogenesis. They are known to promote bud development in
a variety of plants, and to play an important role in the
regeneration and differentiation of organs from callus
tissue. They also elicit various effects on enzymatic and
metabolic activities. The effect of cytokinins on enzyme
activity is an important area of research for it could lead
to a more biochemical description of their behavior and
ultimately to the discovery of their primary mode of action.
•Responses £& stress
One of the most complex areas of cytokinin research-
is the determination of their role in the response of plants
to environmental changes and their interaction with other
hormones to elicit such responses. A more specific area of
research is the determination of their role in the
alleviation of stress. A lot of work has been done to
determine the effects of water stress, high salinity, and
flooding on cytokinin levels. The general trend seems to be
a concurrent reduction in the levels of cytokinins and
proteins, and rate of amino acid incorporation. These
13
responses can be relieved by kinetin treatment (Itai and
Vaadia 1965; Itai, Richmond, and Vaadia 1968; Burrows and
Carr 1969). The general suggestion is that stress induced
reduction of protein synthesis in plants results from a
deficiency of cytokinins. This also applies in the area of
heat stress. Previous work (Cole and Steponkus 1968) has
shown that the roots are responsible for the perception of
high temperatures and that the visual symptoms of heat
stress in a plant are due to a thermally induced deficiency
of a substance which is produced in the roots. This, of
course, leads to a theory which is testable; since
cytokinins are produced in the roots (Weiss and Vaadia 1965
Kende 1965; Kende and Sitton 1967), perhaps they are that
"deficient substance". One may argue that the physiology
involved with heat tolerance is much too complicated for
this theory to have any significance, but by concentrating
on just one facet of the problem perhaps it is possible to
use it as a means of producing more durable plants.
When plants are exposed to various types of stress
their hormone levels undergo significant and rapid changes.
These changes result in the regulation of various
physiological processes such as membrane permeability and
water potential, protein synthesis and degradation,
photosynthesis and respiration, and enzyme regulation.
Various structural changes are also elicited indirectly by
these sudden and dramatic changes. Little evidence is
14
available as to the exact role that hormones play in
response to stress, but as mentioned earlier, a further
understanding of their key mode of action will be required.
In the case of temperature stress, some studies have
been conducted and the response of hormones examined.
Indole acetic acid oxidase activity in winter wheat
seedlings has been shown to increase upon exposure to low
temperatures (Bolduc, Cherry, and Blair 1970). This
increased IAA oxidase level should result in low auxin
levels at low temperatures. Gibberellin levels are thought
to increase under low temperatures since gibberellins are
often employed in lieu of cold treatment to break dormancy
of seeds and to induce flowering. There are no data,
however, which strongly support this hypothesis. Ethylene
production is enhanced after cold stress, and this has been
suggested to occur as a result of an induced phase change in
membrane lipids during chilling and an increase in membrane
permeability (Wright 1974). High temperatures, on the other
hand, cause a decrease in ethylene production (Burg and
Thimann 1959). It appears that the enzymes involved with
ethylene biosynthesis are relatively heat labile. Abscisic
acid levels increase in response to cold or heat stress. It
is not known whether enhanced ABA production is triggered by
the stress conditions themselves or by the water loss that
they cause. Experiments by Hiron and Wright (1973) suggest
that perhaps ABA production is a response to water deficit.
15
Cytokinin levels in roots and xylem have been shown
to increase in response to cold temperatures (Engelbrecht
and Bielinska-Czarnecka 1972; Skene 1972). In contrast,
increases in root temperature lower the cytokinin level
present in the xylem (Skene and Kerridge 1967) as well as in
the leaves and roots themselves (Gur, et al. 1972; Zieslin,
et al. 1984). The lowering of these levels could be due to
a decrease in the synthesis and transport of cytokinins, or
to an interconversion of cytokinins. Whatever the
mechanism, it has been shown that more tolerant individuals
maintain higher levels than nontolerant individuals (Cole
1969).
Extraction. Purification. Identification
Extraction
Cytokinins are extracted from plant tissue with
aqueous ethanol or methanol. Less polar solvents are
usually not employed because of the low solubility of
cytokinins in them. Extractions are frequently carried out
at low temperatures (around -20C) to minimize enzymic or
chemical degradation. It should be emphasized that
extraction at low temperatures or with cold organic solvents
does not inactivate enzymes such as phosphatase very
rapidly. Therefore, it is suggested for those intent on the
study of nucleotide cytokinins, that alkaline phosphatase be
inactivated before extraction. This can be accomplished by
16
using methanol-chloroform-formic acid-water (12:5:1:2 v/v/v)
and the presence of a free radical trap such as BHT or
propyl gallate at -25C (Letham 1966). Extraction methods of
cytokinins from plant material have been reviewed by Horgan
(1978).
Purification
Column chromatography has been an extremely useful
tool in isolating cytokinins. Polystyrene sulphonic acid
resins (dowex) are extensively used for initial purification
of crude extracts on a large scale or in bulk. These
cation-exchange resins are usually used in the protonated or
acid form, although cytokinin bases and ribosides retained
in this form are subject to hydrolysis. Bound cytokinins
are then eluted with ammonium hydroxide, usually around 5N.
Cation-exchange resins are useful for producing cytokinin
rich extracts, but they do not separate or fractionate the
cytokinins into their various chemical forms. The most
widely used column for the fractionation of cytokinins is
sephadex LH-20 (Armstrong, et al. 1969; Hewett and Wareing
1973). High performance liquid chromatography separation of
cytokinins is without a doubt the most valuble technique for
rapid screening of mixtures. Its successful use has been
reported from various laboratories (Pool and Powell 1974;
Carnes, Brenner, and Andersen 1974; Challice 1975). Coupled
with radioimmunoassay, HPLC has proven a powerful technique
for cytokinin identification and quantitation (HacDonald,
17
Akiyoshi, and Morris 1981).
Paper and thin layer chromatography have been used
to purify cytokinins. In fact, all naturally occurring
cytokinins known can be separated by TLC methods. Usually
silica or cellulose gel layers are employed. Sometimes more
than one solvent system may have to be used in order to
resolve very complex mixtures, but closely related compounds
are none the less separable (Letham, et al. 1977).
Identification
If cytokinins have been isolated in sufficient
amount and in pure enough form, then techniques such as
nuclear magnetic resonance, infrared spectroscopy,
ultraviolet spectroscopy, and mass spectrometry can be used
for identification. Spectra of unknown samples are compared
with authentic or standard samples for identification, or a
mixed melting point test can be conducted. Usually
cytokinins cannot be obtained in sufficient quantity or in
pure enough form for identification. Other analytical
methods, such as paper chromatography, TLC, GLC, GLC-MS,
HPLC, and HPLC-radioimmunoassay are used for the
identification of plant hormones present in minuscule
amounts.
Bioassays
Bioassays were initially developed as a means for
establishing the presence of endogenous plant growth
18
substances and to facilitate in their isolation. They have
also been used as a way of analyzing the types of hormones
present in an extract. Their limitations in this role are
well known, and present physiochemical methods are more
reliable. Furthermore# even though bioassays are relatively
selective for a particular hormone in comparison to
physiochemical methods, no one assay system known today is
free from interaction with impurities present in the
extracts. Thus, even though a bioassay renders itself
repeatable, the accuracy is always questionable. Taken in
conjunction with more definitive techniques, bioassays can
serve as a means for the verification of the presence of a
particular compound as judged by activity. For example, if
one is isolating cytokinins, one way of making sure that the
compound extracted is indeed a cytokinin is to check it with
one of the bioassays specific for cytokinins. Furthermore,
if one is trying to determine the physiological effects of a
hormone such as a cytokinin, it is a necessity to check that
the compound isolated exhibits biological activity. Two
relatively quick and easy bioassays which are also fairly
reliable in detecting cytokinin activities are the barley
leaf senescence bioassay (Kende 1964), and the radish
cotyledon bioassay (Letham 1968). Neither are recommended
for quantitative work, but they, are fairly useful for crude
approximations or for comparative analyses. Table 3 lists
some of the more commonly used bioassays, their
Table 3: List of commonly used bioasBays
BIOASSAY ASSAY TIME LOWEST DETECTABLE REFERENCE (DAYS) CONC. OP KINETIN
t u g / 1 )
bean leaf disc expansion 2 200 Miller (1956, 1963)
Lemna minor (dark growth) 2 60 Hillman (1957)
tobacco stem pith 21 40 Bottomley et al. (1963)
Xanthium cotyledon expansion 4 20 Esashi and Leopold (1969)
Anaranthus betaeyanin 1-3 2-2 Bigot (1968)
radish cotyledon 3 10 Letham (1968, 1971)
radish leaf expansion O.B 2-10 Kuraishi (1959)
oat leaf senescence 4 3-10 Thimann and Sachs (1966)
barley leaf senescence 2 3 Kende (1964, 1965)
soybean callus 21 1 Miller (1963)
cucumber cotyledon 0.8 1 Fletcher and McCullagh (1971)
carrot phloem 21 0.5 Letham (1967)
tobacco callus 35 <1 Murashige and Skoog (1962)
20
sensitivities/ and the amount of time required for each.
High temperature effects £H alfalfa
The effects of heat stress on alfalfa (Medicago
sativa L.) have been the subject of study for many
researchers. Heat stress invariably causes a reduction in
the yield and quality of alfalfa. In the southwestern
United States, a decline in forage production during the
summer months, when minimum temperatures are high, limits
forage yield. This phenomenon, known as "summer slump"
(Robison and Massengale 1967, 1968; Foutz 1973), has been
studied in order to find a way of reducing its impact.
It has been found that the decrease in forage yield
results from a reduction of growth in response to high
temperatures (Pulgar and Laude 1974). McDaniel, et al.
(1981) have linked this growth reduction with loss of carbon
due to uncontrolled high plant respiration rates at these
high temperatures. The period of growth depression
increases as the intensity of heat stress is increased; for
example, as the temperature increases or as the time of
exposure to high temperatures increases (Pulgar and Laude
1974). It is unclear as to when heat stress begins to exert
its effects on growth. It is known that the application of
heat stress at one stage in development affects growth at a
later stage (Laude 1964), but it seems unlikely that this
type of response is the cause of the growth declination
21
observed during the "summer slump". Ku and Hunt (1977) have
shown that factors which enable plants to respond to a
uniform temperature regime differ from those that enable
plants to respond to rapid temperature changes. The "summer
slump" is probably a result of this rapid temperature change
response, since the decline in forage production occurs at
the same time that night temperatures increase. The exact
mechanism of this reduction in growth has not been
determined. There are several physiological factors which
are known to occur as a result of heat stress or increased
temperatures which may somehow act together to elicit the
overall declination in growth. To begin with, at high
temperatures photorespiration is greater than it would be at
low temperatures, and the rate of carbon dioxide exchange is
decreased (Ku and Hunt 1977). This, coupled with the facts
that leaf area indices decrease with increasing minimum
temperatures (Robison and Massengale 1968), and that alfalfa
plants growing under high temperatures are ready to harvest
before enough photosynthates have been produced (Robison and
Massengale 1967), results in a decrease of carbohydrates in
the roots (Robison and Massengale 1967, 1968; Feltner and
Massengale 1965). This decrease in the reserves needed for
growth results in various physical alterations such as the
reduction in shoot numbers and length (Pulgar and Laude
1974; Robison and Massengale 1967), in the number of
internodes and nodes (Robison and Massengale 1967), in the
22
leaf area indices thus leading to a reduction of
photosynthesis (Ku and Hunt 1977), and the whole process
continues to cycle. The final result is inevitable; a
reduction in plant growth and crop production.
MATERIALS AND METHODS
Plant Material
Two strains of Medicaao sativa L. were used as a
source of plant material: cv. *Lew' and Low
Photorespiration. Plants were provided by Dr. A. K. Dobrenz
of the University of Arizona. A field of approximately 100
selected individuals was planted in the summer of 1983. The
plants were allowed to grow under field conditions, and
samples were taken from each strain. Plants were harvested
every two to three weeks during the sampling period.
Sampling
Individual plants were harvested about 2 to 3 weeks
after a mowing to ensure the presence of young growing
tissue. Usually a whole plant was cut and immediately
placed on ice. Every leaf was picked from its stems while
taking precautions to keep the plant material frozen. The
leaves were then kept frozen at -70C until they were needed
for extraction. One plant, on the average, would yield
about 100 g of leaf material.
Extraction procedures
The first extraction procedure employed was one
described by Prank Cole (1969). Frozen leaves (0.5 g) were
extracted in 2.0 ml citrate-phosphate buffer (0.18M), pH
7.2, at 25C. The extraction consisted of grinding up the
23
24
leaf material with a glass rod in the buffer solution and
then centrifuging at 271 a's (1500 r.p.m.) for 5 minutes.
The supernatant was collected and acidified to a pH of 2.5
with IN HC1, and poured onto a cation exchange resin (Dowex
50W-X8, 100-200 mesh), which had been protonated with 10%
HC1 (v/v) and washed to neutrality with distilled and
deionized water. The bound material was eluted off the
column at room temperature with 10 ml of 10% NH^OH Cv/v)
followed by 10 ml of distilled and deionized water. The
combined NH4OH and water washed elutants were lyophilized on
a Lab Con Co. freeze drier and the residues were stored
frozen until analyzed.
The second extraction procedure used was one
developed by van Staden and Davey (1977) for extraction of
cytokinins from callus tissue. Frozen leaves CI g) were
homogenized in 5 ml of 80% (v/v) ethanol using a polytron at
a speed setting of 3 for 30 sec and then stirred for 48 h at
room temperature. The extract was then filtered on a
Buchner apparatus with no. 2 Whatman filter paper and
reduced to dryness in vacuo at 35C using a Buchi rotovac.
The residue was redissolved in 5 ml of 70% ethanol, the pH
adjusted to 2.5 with 0.1N HC1 , and the resulting solution
passed through a cation exchange column (Dowex 50W-X8;
Hydrogen form; 200-400 mesh; 20 g). The column was washed
with 5 ml of distilled water followed by 10 ml of 70%
ethanol (v/v). These two elutants were combined, taken to
25
dryness in vacuo at 35C by rotoevaporation, and stored
frozen until analyzed. The bound substances were eluted
from the column with 25 ml of 5N NH^OH. The ammonia was
removed by rotoevaporation at 35C and the residue stored
frozen until analyzed.
The third extraction procedure used was one
developed by Letham and modified by Mauk (1983). Frozen
leaf tissues (10 g) were suspended in 10 ml of extraction
medium I (MeOHjCHC^jHCOOHil^O 12:2:1:2 v/v and 20 mg
propyl gallate) and allowed to sit overnight in a freezer at
-20C. The tissue solutions were then homogenized using a
polytron at a speed setting of 4 for 30 sec, the polytron
was rinsed with 5 ml of extraction medium I which was
combined with the other 10 ml, and the resulting 15 ml were
centrifuged at 271 a's for 20 min at 5C. The supernatants
were collected and stored in a freezer (-20C). The pellets
were resuspended in 15 ml of extraction medium II
(MeOH:HCOOH:H20 6:1:1 v/v and 20 mg propyl gallate) and
centrifuged again at 271 s's for 20 min. These supernatants
were then collected and combined with the previously
collected ones. The pellets were discarded. The combined
supernatants were concentrated to about 1 to 2 ml by
rotoevaporation at 35C, the pH adjusted to 3.0 with 0.1N
HC1, and passed through a cation exchange column (Dowex 50W-
X8; Hydrogen form; 200-400 mesh). After a wash of 10 ml
with distilled and deionized water, the bound substances
26
were eluted with 10 ml of 5N NH4OH. The ammonia was removed
by rotoevaporation and the residue stored frozen until
analyzed.
The last extraction procedure utilized is a
modification of Mauk (1983). Frozen leaf tissue (100 g) was
suspended in a beaker in enough extraction medium I (same as
in Mauk procedure) to cover the leaves, and allowed to sit
approximately 24 hours at -20C. The tissue solution was
poured into a Wareing blender and homogenized for 1 to 2 min
at low speed setting. The resulting extract was poured into
about 18 to 20 15 ml centrifuge tubes and centrifuged at 271
a's for 20 minutes at 5C. The supernatants were collected
and stored in a freezer. The pellets were resuspended in 15
ml of extraction medium II (same as in Mauk procedure) and
centrifuged again at 271 a's for 20 minutes. These
supernatants were collected and added to the previous ones.
The combined supernatants were reduced to dryness by
rotoevaporation at 35C. The resulting residue was
resuspended in 10 ml of distilled and deionized water,
passed through a no. 2 Whatman filter using a Buchner funnel
and vacuum. The filtrate was then passed through a 0.22 #iM
millipore filter, again using a vacuum to facilitate the
process. This resulting filtrate was adjusted to a pH of
3.0 with HC1, and poured through a cation exchange column
(Dowex 50W-X8; pH 3.0 with IN HC1; 200-400 mesh). The
initial elutant was saved, reduced to dryness, and stored
27
frozen. The column was washed with 20 ml of deionized and
distilled water which was collected, reduced to dryness and
stored frozen. The bound substances were then eluted with
20 ml of 5N NH^OH followed by 20 ml of distilled and
deionized water. These combined elutants were also reduced
to dryness by rotoevaporation at 35C and stored frozen.
Table 4 summarizes the four extraction procedures
based on the extraction media and the amount of leaf tissue
used in each.
Testing q£ Extracts
Ultraviolet Spectroscopy
All of the frozen residues from each of the
extraction procedures were resuspended in 2.0 ml of pure
methanol and filtered with a 0.22 pM millipore filter to
remove any particulate matter. Each of the samples required
diluting in order to obtain a u.v. spectrum. In most of the
cases this dilution consisted of 1.0 ml from the above 2.0
ml added to 9.0 ml anhydrous HeOH. Some of the extracts
from the fourth extraction procedure required double and
triple dilutions. Table 5 lists the extracts and their
dilution factors. The spectra were recorded over a range of
wavelengths from 300 nm to 220 nm with the maximum set at
268 nm corresponding with that for zeatin. The results were
taken as an indication of the relative amounts of cytokinins
(zeatin) and other u.v. absorbing compounds present in .each
extract.
Table 4: Summary of extraction procedures used based on leaf tissue amount and the type of extraction medium used.
Extraction Procedure
GramB of Leaf Tissue
Extraction Medium
1 0.5 Citrate Phosphate Buffer (0.18M) pH
2 1.0 EtOH
3 10 MeOHsCHCl,:HCOOH:H HeOH:HCOOH:H20 (6:!
5O (12:2:1:2 v/v) 1:1 v/v)
4 100 MeOH:CHC13:HCOOH:H MeOH:HCOOH:H->0 (6:
20 (12:2:1:2 v/v) 1:1 v/v)
29.
Table 5: List of sample eluants and their dilution factors.
Extraction Procedure
Sample Eluant Dilution (mis sample:mls MeOH)
original H2O/NH4OH original H2O/NH4OH
1:9 1:9 1:9 1:9
original H20/Et0H NBJOH original H20/Et0H NH4OH
1:9 1:9 1:9 1:9 1:9 1:9
original H2° NH4OH original B20 NB4OH
1:9 1:9 1:9 1:9 1:9 1:9
original H20 NB4OH original H20 NH4OH
1:29 1:19 1:19 1:29 1:19 1:19
Lew
Low Photo
Lew
Low Photo
Lew
Low Photo
Lew
Low Photo
original signifies the eluant after the extract had been passed through the column and recollected. Similarly, H20/NH40H, H20, and NH4OH signify the eluants by the type of wash employed in obtaining them.
30
Thin Layer Chromatography
Five to ten 1 #*1 drops of each methanolic
resuspension of the extracts were spotted on premade 20 x 20
cellulose P thin layer chromatography plates (Baker Co.) and
developed to 15 cm in a tank containing 100 ml of 60%
methanol (v/v). Only the extracts whose u.v. spectra
indicated the presence of cytokinins were subjected to TLC
for further analysis. Rf values were calculated and the
spots were scraped from the plates and suspended in 3 ml of
buffer for use in a bioassay. Spots were detected using a
shortwave ultraviolet lamp.
Bioassays
Barley Leaf Senescence BlQassay Barley seeds were
germinated in trays which contained germination paper wetted
with 40 ml of distilled water. The trays were placed in a
dark chamber at 26C for 48 h. The seedlings were then
planted in potting soil in trays and placed in a growth
chamber set for a 16 h photoperiod, and a day/night
temperatures of 20/17 +/- 2C. After two weeks, barley
seedlings were selected for uniform size and a 1 cm segment
3 cm from the top of the blade, was excised from each
seedling harvested. Four leaf segments were placed in each
of four petri dishes per treatment (16 segments per
treatment) containing 3.0 ml of citrate phosphate buffer
(0.18M) pH 7.2, or 3.0 ml citrate-phosphate buffer
31
resuspended residues from TLC plates, pH 7.2. Dishes were
kept in the dark for 48 h after which the segments from each
one were boiled in 80% ethanol (v/v) with boiling chips to
extract the chlorophyll. The chlorophyll extracts were
analyzed on a Beckman spectrophotometer at 665nm. The
absorbancies obtained were used as an indication of the
relative amounts of chlorophyll present in each of the petri
dishes. Higher absorbancies would signify higher levels of
chlorophyll which in turn would indicate a slower senescence
rate denoting the presence of cytokinins. . The samples which
show a greater retention of chlorophyll are then assumed to
possess higher levels of cytokinins.
Radish Cotyledon Bioassay Radish seed (cv. Scarlet
Globe1) were germinated at 26C in darkness in petri dishes
on well wetted Whatman no. 2 filter paper. After at least
30 h, the smaller cotyledon was excised from each seedling,
and great care was taken to remove the hypocotyl completely.
Cotyledons (8 to 12) were weighed in separate groups, and
then distributed so that each group had the same average
weight.
The extracts scraped from the TLC plates were
resuspended in 3.0 ml of 2 mM phosphate buffer pH 5.8 to
6.0. Filter paper circles (Whatman No. 2, diant. 9 cm) were
wetted with 3 ml of cytokinin solutions (from TLC
scrapings), or with 3 ml phosphate buffer (2 mM) pH 5.8 to
6.0 (these served as the controls). The cotyledons
32
carefully weighed out previously were then added. After 4
to 5 days under fluorescent lighting, average weights of the
cotyledons in each dish were taken and compared with their
initial weights.
High Performance Liquid Chromatography
The diluted samples which gave a positive indication
for the presence of cytokinins based on the ultraviolet
spectra were then subjected to further analysis. The
technique employed was reverse phase high performance liquid
chromatography on a Waters Inc. HPLC unit consisting of a
dual pump system with proportioning control, an integrator
and a u.v. detector set at 254 nm. Samples ( 25 pi ) were
applied to a 250 x 4.6 mm, 5 /am spherical octadecyl column
(Alltech) and eluted with 60% methanol over a period of 30
minutes at a flow rate of 1.5 ml per minute. The eluting
solvents were vacuum degassed prior to pumping through the
column in order to avoid the formation of air bubbles in the
system. It was found that some of the samples could be
eluted in fifteen minutes.
The only purine that was run as a cytokinin
reference standard was zeatin, since it was the one of
interest. Each sample was spiked with an equivalent of 5.5
Mg of zeatin. Comparison of the peaks obtained from a
sample with those of the standard containing the same
amount of zeatin that each sample was spiked with lent a
general idea as to the presence or absence of zeatin in the
33
sample. The error involved due to the spiking process was
determined by adding 0.25 ml of 0.001M zeatin to three
separate vials each containing 0.75 ml MeOH. These were
then subjected to analyses on the HPLC. The area of the
peaks for each of the solutions was calculated and an
analysis of error based on area differences was determined.
Variation in retention times of the peaks due to changes in
resin packing, solvents, and samples were also noted, but a
stringent analysis of the error involved was not determined.
The last source of variation taken into account was the
change in peak areas from day to day. This was determined
by injecting the standard each day before running the
samples. The change in peak area for the standard from day
to day was used to calculate a mean area to which all
samples were compared.
RESULTS AND DISCUSSION
Ultraviolet Absorption Spectra
The ultraviolet spectrum of a compound arises from
the absorption of ultraviolet radiation by the molecule
resulting in the transfer of that molecule to a higher
energy state. This high energy transformation is
characterized by the transfer of electrons from lower
orbitals to higher orbitals in the molecule. In order to
absorb in the near ultraviolet range a molecule must exhibit
a rather stable excited state. Usually only unsaturated
molecules exhibit this property. Strong absorption in the
near ultraviolet region occurs when two or more unsaturated
molecules are conjugated to allow interaction of the pi
electrons (pi-pi conjugation). A shift in wavelength as
well as a change in the intensity of absorption will occur
when a multiple bond is conjugated to an atom carrying a
lone pair of electrons (pi-p conjugation). Thus,
ultraviolet spectroscopy is used for the identification of
conjugated groups. The structures of cytokinins, some of
which were shown in figure 1, contain the conjugated double
bond system necessary to allow absorption under ultraviolet
wavelengths. Unfortunately other compounds such as
phenolics, adenine and amino acids also contain structures
34
35
which allow them to absorb in the ultraviolet range, and one
must keep this in mind when analyzing samples thought to
contain cytokinins. The absorption spectrum for zeatin is
represented in figure 2. It shows an absorption maximum at
270 nm and a minimum at 253 nm. The u.v. spectra for all of
the samples will be compared to that for zeatin as a general
way of determining whether zeatin-like compounds are present
in any of the extracts.
Absorption spectra for all of the extracts are
represented in figures 3 through 10. Figures 3 and 4
represent the scans for extracts of the Lew and Low
Photorespiration strains obtained by extraction procedure
one, respectively. The spectra labeled A (original) are
those of the extracts after" they had been passed through a
cation exchange resin and recollected. The absence of any
prominant absorption peaks indicates the retention of all
ultraviolet absorbing compounds present by the column.
These substances were then washed from the column and are
represented in the spectra labeled B (NH4OH/H2O). These
spectra clearly indicate the presence of some type of
conjugated double bond system with a maximum absorption at
269 nm. To further elucidate the nature of the absorbing
compounds, the samples which showed absorption under
ultraviolet wavelengths were then subjected to analysis by
reverse phase high performance liquid chromatography (RP-
HPLC).
ZEATIN
u-
ti
ll 2tO
Figure 2: Ultraviolet spectrum for zeatin.
37
LEW El
90 1 22o '""33 1 3o 1 3So ' no
X, mp
Figure 3: Ultraviolet spectra for eluants of the Lew cultivar obtained by extraction procedure one. A) original extract B) NH4OB/H20 wash.
38
LOW PHOTO El
Figure 4: Dltraviolet spectra for eluants of the Low Photo cultivar obtained by extraction procedure one. A) original extract B) NH4OH/H2O wash.
39
Figures 5 and 6 represent the scans for the Lew and
Low Photorespiration strains obtained by extraction
procedure two, respectively. Again the spectra labeled A
(original) indicate the absence of any absorbing compounds.
The spectra labeled B. (H20/EtOH)r however, do show the
presence of u.v. absorbing compounds with maxima at 269 nm,
wheras the spectra labeled C (NH^OH) do not. These last two
sets of spectra were not as expected. It was hoped that
perhaps the cytokinins present in the extracts would bind to
the columns strongly enough to be removed only by NH^OH.
Either the columns used were not efficient enough to retain
the cytokinins or perhaps the pH values of either the
columns or the extracts did not remain stable. On the other
hand, it could be that the substances yielding the
absorption spectra are some other conjugated double bond
system such as adenine or phenolics. One would then expect
the NH4OH washes to yield an absorption spectra based on the
presence of cytokinin like substances. The fact that these
were not obtained could be explained on the basis that only
1.0 g of leaf tissue was used in this extraction procedure.
This may not be enough to yield any detectable amounts of
cytokinins, or more specifically zeatin. Once again, those
extracts showing absorption under ultraviolet wavelengths
were subjected to further analysis by RP-HPLC.
Figures 7 and 8 represent the scans for the Lew and
Low Photorespiration strains obtained by extraction
40
14-LEW E2
u-
i>
Il
ls-
M-
200 300
\mn
Figure 5: Dltraviolet spectra for eluants of the Lew cultivar obtained by extraction procedure two. A) original extract B) H20/Et0H wash C) NH4OH wash.
41
E!b "fTwI
LOW PHOTO E2
Figure 6: Ultraviolet Bpectra for eluants of tbe Low Photo cultivar obtained by extraction procedure two. A) original extract B) H20/EtOH wash C) NH40B wash.
42
LEW E3
% 1 in 1 jl« 1 i, 1 io ' MO
X,m (jl
Figure 7: Ultraviolet spectra for eluants of the Lew cultivar obtained by extraction procedure three. A) original extract B) B2O wash C) NH4OH wash.
43
U-
12*
E,mu •"TTWi
M-
OB-
14-
12-
M
LOW PHOTO E3
Figure 8: Ultraviolet spectra for eluants of the Low Photo cultivar obtained by extraction procedure three. A) original extract B) H20 wash C) NH^OH wash.
44
procedure three, respectively. Again, neither the original
or the NH4OH spectra (A or C) show any indication of the
presence of cytokinin like compounds. The H20 spectra (B)
do, however, show the presence of a conjugated double bond
system with absorption maxima at 270 nm and 273 nm for Lew
and Low Photo respectively. As with the last extraction
procedure, it is not clear as to whether the column
efficiencies are not appropriate, the pHs are off, or the
amounts of cytokinins extracted are not detectable. Samples
showing absorption spectra were subjected to RP-HPLC for
further analysis.
Figures 9 and 10 represent the scans for Lew and Low
Photorespiration obtained by extraction procedure four,
respectively. All three spectra: original (A), H2O (B),
and NH4OB (C) for each of the cultivars show significant
absorption under ultraviolet wavelengths. The spectra
obtained from the original extracts after they had been run
through the columns indicated an inefficient retention of
the u.v. absorbing compounds present in the extracts. This
was most likely a result of overloading the columns since
100 g of leaf tissue were used in this extraction procedure.
This would also explain the absorption spectra obtained from
the NH4OH washes. In other words, enough tissue was used to
obtain cytokinins in detectable amounts. Again the presence
of absorbing compounds in the H20 washes occurs. Further
analysis by RP-HPLC should elucidate the nature of these
45
IS*
U-
eE:
LEW E4
Figure 9: Ultraviolet spectra for eluants of the Lew cultivar obtained by extraction procedure four. A) original extract B) H20 wash C) NH4OH wash.
46
11
14-
12-
LOW PHOTO E4
2^ Tmax
u-
u-
%A
Ba
ll 280
X,nrvi
Figure 10: Ultraviolet spectra for eluants of the Low Photo cultivar obtained by extraction procedure four. A) original extract B) H2O wash C) NB4OH wash.
47
compounds. Table 6 summarizes those eluants which elicited
prominant u.v. spectra and which will be analyzed further by
RP-HPLC.
Since column inefficiency and pH instability were
possible explanations for some of the spectra obtained, a
brief explanation of each is appropriate. Cation exchangers
are substances capable of taking up water and liberating
cations by electrolytic dissociation. The cations set free
may then be substituted by other cations present which
possess a greater affinity for the exchanger. The exchanger
used in these experiments is commercially known as Dowex
50W-X8 which is of the hydrogen form, has a styrene
polymeric matrix, and sulphonic acid as the functional group
or exchange group. The process of cation exchange is
expressed by the equation shown in figure 11. It is a
reversible process whose direction depends not only on the
affinity towards the ion exchanger, but also on the
concentration of the ions present. For example, although a
cytokinin may have a greater affinity for the exchanger than
a proton, it may be readily substituted reversibly by the
proton if the concentration of the latter in the solution is
substantially greater.
The pHs of the column resin and the solution to be
run through the column are important because they
determine whether the column exchanger is in the
protonated form to allow cationic exchanges to occur and
48
Table 6: List of alfalfa leaf eluants subjected to BPLC.
Extraction Sample Eluant Procedure
1
2
3
4
Lew Low Photo
Lew
Low Photo
Lew Low Photo
Lew
Low Photo
H2O/NH4OH B2O/NH4OB
original H20/EtOB original B20/NB40H
H2O
original B20 NH4OB original H20 NH4OB
49
ft-SO-'
" 0> RS0ji5-R + y-t
r«5V
V
Figure 11: Equation expressing cation exchange between functional group of column (R-SO3H) and a cytokinin.
50
whether the competing cations (cytokinins) are also in the
correct form. The pHs of 2 to 3 selected for these
experiments were based on the fact that zeatin is known to
have pKa values of 4.4 and 9.8 in water (Letham et al.,
1964, 1967). Keeping the pH of the solution and the column
below these pKa values ensures the presence of deprotonated
amine groups and protonated sulphonic groups needed for
exchange. Inefficient binding of the exchange cations to
the resin could be due to several reasons. One has already
been mentioned and occurs when the exchange cation is not
present in high enough concentrations. The result is that
the equilibrium of the equation in figure 11 tends to shift
towards the left. In essence, the exchange cation will wash
right through the column with little or no ionic
interaction. Another explanation for inefficient binding
could be that the degree of cross linking of the ion
exchanger may be too high, resulting in small pore sizes
which do not permit the entrance of the substances into the
ion exchanger. The resin used in these experiments has a
cross linking of X8 which is suitable for small inorganic
and organic molecules such as amino acids. Cytokinins fall
into this category and should exchange quite nicely on this
resin. Perhaps the granulation of the ion exchanger was too
coarse or too fine to allow equilibrium to be attained
between the mobile and stationary phase during the flow
through the column. This was kept in mind during the course
51
of the experiments using a mesh of 100-200 when the extracts
were fairly small and dilute, and using a mesh of 200-400
when the extracts were larger and more concentrated. The
flow rate was kept fairly stable at around 1 to 2 mis per
minute and should have been sufficient to allow equilibrium
to be attained. Perhaps the bed volume of the resin was not
large enough to accomodate the sample, but this would not
serve as an explanation in the cases where sample sizes were
both small and dilute. The only other explanation appears
to be in the pH stability of the column. The pH of the
samples was fairly easy to adjust and remained constant
under the emperimental conditions employed. That of the
column, however, was difficult to adjust and would show
variation with time.
Reverse Phase High Performance Liquid Chromatography
Those samples which revealed a strong ultraviolet
absorption spectrum were reasoned to contain some type of
conjugated double bond system analagous to that found in
cytokinins. The u.v. spectra were used as a preliminary
screening procedure for the detection of the presence of
cytokinin like substances. It is for this reason that no
quantitative analyses of the spectra utilizing known
absorption coefficients for some of the cytokinins were
attempted. Instead, those samples yielding a significant
u.v. spectrum were subjected to analysis by reverse phase
52
high performance liquid chromatography. The chromatograms
obtained from these samples are represented in figures 12 to
17.
Only those peaks which show an elution time similar
to that for zeatin were analyzed in detail. Although other
peaks were present in some of the chromatograms, their
identification would depend on the availability of several
other cytokinin-like standards such as isopentenyladenine
and its riboside, zeatin riboside, adenine, adenosine, and
perhaps some related compounds such as xanthine and
hypoxanthine. In order to keep this project simple and
direct, the identification of other peaks present in each
chromatogram will be attempted at a later date. The
objective at hand, however, is to determine whether zeatin
has been extracted and isolated from alfalfa leaves.
The results of the error analyses done for the
spiking process as well as for the day to day variation are
shown in table 7. A mean value for the peak area of 5.5 mg
of zeatin was determined to be 0.702 cm^ +/- 0.083. The
variation due to spiking was +/- 0.045 which increases the
peak area range. In other words, any peak area falling in
the range of 0.657 cm^ to 1.07 cm^ will not be considered
significantly different from the area due to the 5.5 ng of
zeatin each sample was spiked with. If, however, a peak
shows a significant increase in area from that of the
53
Table 7: Results of error analyses for a) area differences from day to day and b) area differences due to Bpiking.
a) Area (cm2)
Day I 0.800
Day II 0.645
Day III 0.743
mean 0.702 variance 0.083
b) Area (cm2)
Spike I 0.743
Spike II 0.658
Spike III 0.673
mean 0.691 variance 0.045
54
standard, then it will be concluded that zeatin has been
successfully extracted from that sample. Microgram (or
milligram) equivalents have been calculated for these
samples using a peak area ratio with the standard and the
total amount of zeatin extracted from these samples has also
been calculated. Table 8 summarizes the dilution factors
for the samples subjected to RP-HPLC.
Figure 12 represents the chromatograms for tissue
extracts of the two cultivars obtained by extraction
procedure one. The slowest eluting peak in each
chromatogram is zeatin and in neither case is the area
significantly different from that of the standard. Since
only 0.5 g of leaf material were used in this extraction
procedure, one could argue that not enough material was used
to extract detectable amounts of zeatin. Also, the lack of
an oxidase inhibitor in this system could have allowed the
breakdown of zeatin. Indeed, the other peaks present in the
chromatograms could be such breakdown products. To test
this, one could conduct a study in which a solution of
zeatin is allowed to react with its oxidase and the products
analyzed by RP-HPLC. A comparison of the chromatograms
obtained with those from extraction procedure one could then
determine whether the other peaks present are indeed
breakdown products of zeatin. If they are not then further
identification of these peaks would be determined by running
other standards and comparing the chromatograms.
55
Table 8: Dilution factors for samples subjected to BPLC.
Extraction Sample eluant Dilution Procedure
1 Lew Low Photo
B9O/NH4OB B2O/NB4OH
1:9 1:9
2 Lew
Low Photo
original' B20/EtOH original
1:9 1:9 1:9
3 Lew Low Photo
H?0 H2O
1:9 1:9
4 Lew
Low Photo
original B2O NB4OB original H2° NH4OH
1:19 1:19 1:19 1:19 1:19 1:19
56
<3-iO CVJ <
275 RT (MIN)
<3-to OJ <
w
2.77
Figure 12: BPLC cbromatograms for the eluants of the two cultivars obtained by extraction procedure one. Top: Lew cultivar NHAOH/EtOH wash Bottom: Low Photo cultivar NH^OB/EtOH wash. The abscissa • indicates retention time in minutes.
57
Figures 13 and 14 represent the chromatograms for
tissue extracts of both cultivars obtained by extraction
procedure two. Again, the slower eluting peak is zeatin.
In neither case, for either of the washes, was a significant
amount of zeatin detected. The Low Photo eluant labeled
ORIGINAL yielded a rather confusing chromatogram. It
appears that there are other contaminating compounds present
probably due to an insufficient clean-up of this sample.
That these peaks are a result of the presence of other
cytokinin like compounds, however, cannot be ruled out. Due
to the ambiguity involved, the peak area for the added
zeatin was not calculated. It seems unlikely that a
significant amount of zeatin would be found in this wash
after it had been run through an exchange resin.
Figure 15 represents the chromatograms for tissue
extracts of the two cultivars obtained by extraction
procedure three. In both cases the slowest eluting peak is
zeatin. The Lew extract did not yield a significant amount
of zeatin, whereas the Low Photo extract did. The ratio of
the peak area for the Low Photo extract to the peak area for
the 5.5 jig zeatin standard is 3.33. This number, multiplied
by 5.5 fig, gives the relative amount of zeatin present in
the injected sample, which in this case turns out to be
18.32 Mg* Subtracting 5.5 fig gives the amount of zeatin
present in the unspiked extract. Now in order to take the
dilution factors from spiking into account this number is
58
2.78 RT (MIN)
2.69 RT (MIN)
Figure 13: HPLC chromatograms for eluants of the Lew cultivar obtained by extraction procedure two. Top: ORIGINAL eluant Bottom: HpO/EtOH wash. The abscissa indicates retention time in minutes,
59
RT (min)
m csj
2£9 RT (min)
Figure 14: BPLC chromatograms for eluants of the Low Photo cultivar obtained by extraction procedure two. Top: ORIGINAL eluant Bottom: B20/EtOH wash. The abBcissa indicates retention time in minutes.
60
ir» csi
J^AJ <
RT (min)
in CM
RT (min) 15: HPLC chromatograms for eluants of both cultivars obtained by extraction procedure three. Top: Lew HoO wash Bottom: Low Photo H2O wash. Tne abscissa indicates retention time in minutes.
61
then divided by .75 ml and then multiplied by 1.0 ml,
yielding 17.07 ng. Now the dilution factor mentioned in
table 8 is taken into account by dividing 17.07 by 1.0 ml
and then multiplying by 10 ml to give 171.0 ftg. Since only
half of the original sample was used in making the dilution,
this number is then multiplied by 2 to give a grand total of
342 pg of zeatin present in this extract.
Figures 16 and 17 represent the chromatograms for
the two cultivars subjected to extraction procedure four.
In the chromatogram labeled ORIGINAL of figure 16, the
zeatin peak is the largest one present, or the slowest
eluting one. It represents an equivalent of 18.8 fig of
zeatin. It has been found that this eluant contains a
calculated equivalent of 709 fig of zeatin, indicating that
the majority of the zeatin in this eluant did not bind to
the column resin. Since 100 g of leaf tissue were used in
this extraction procedure, it is possible that the increased
concentration of zeatin in the extract overloaded the
capacity of the column. One would need to increase the bed
volume of the column in order to handle such rich extracts.
In the chromatogram labeled H20 of figure 16, the
slowest eluting peak, or the second peak of the pair, is the
one whose retention time most closely resembled that for the
zeatin standard. Its area represents the equivalent of 13.2
Mg of zeatin, and the total amount present in this eluant was
calculated to be 411 Mg- This again shows that the columns
s CM
IF RT (mm)
m N
V
756 RT (min)
s (M
<
RT(min)
Figure 16: HPLC ChromatogramB for eluants of the Lew cultiva£ obtained by extraction procedure four. Leftz ORIGINAL Top right: H2O wash Bottom right: NH4OH wash. The abscissa indicates retention time in minutes.
63
157 RT(min)
in CM
3 56 RT (min)
RT (min) Figure 17: HPLC chromatograms for eluants of the Low Photo
cultivar obtained by extraction procedure four. Top: ORIGINAL Middle: B^O wash Bottom: NH4OH wash. The abscissa indicates retention time in minutes.
64
are not binding as well as they should be.
In the chromatogram labeled NH4OH of figure 16,
again the slowest eluting peak is the one corresponding to
zeatin. Its area was calculated and used to determine the
presence of 9.7 tig of zeatin, which amounted to a total of
224 fxg from this eluant. According to the results for the
Lew cultivar, a minimum total of 1.3 mg equivalents of
zeatin were extracted. Only about 1/6 of this was actually
retained on the column resin, the rest coming through the
column, or being washed off with water. It appears that the
extraction procedure is satisfactory, but optimization of
the column system is required. In the chromatogram labeled
ORIGINAL of figure 17, the zeatin peak is the slowest
eluting one. It represents an equivalent of 11.7 ng of
zeatin. The total amount present in this eluant was
calculated to be 331 Mg.
The slowest eluting peak in the chromatogram labeled
H2O of figure 17 is the peak corresponding to zeatin. It
represents an equivalent of 8.4 /ig of zeatin. The total
amount calculated for this eluant was 155 ng .
In the chromatogram labeled NH4OH, the zeatin peak
is again the slowest eluting one. Its peak area renders an
equivalent of 14.8 ng of zeatin present. The total for this
eluant was calculated to be 496 ng. The results indicate
that a minimum total of 0..98 mg equivalents of zeatin were
extracted from Low Photo. The same problems with column
65
efficiency are indicated in the results of extraction
procedure four on Low Photo, although to a lesser degree. A
lot of the zeatin is eluting through the column without
binding, but more is coming off in the NH^OH wash (1/2 of
the total) than in the H2O wash. This is by no means
indicative of a more efficient system, but it shows that
column efficiency can be improved for this system by
increasing its capacity for the extracts either by
monitering pH more stringently, increasing the bed volume,
or by limiting the amount of extract run through a column at
one time. Differences in the results for extraction
procedure four between the two cultivars may also be
explained on the basis of inefficient column work. Although
inherent differences due to the nature of the cultivars
cannot be ruled out, an attempt to reduce the variability by
optimizing the column system should be considered.
The results for the eluants subjected to HPLC are
summarized in table 9. Differences between the four
extraction procedures are evident. Clearly, the more tissue
used in the extraction procedure, the more zeatin is capable
of being extracted. This is demonstrated by the fact that
extraction procedure four yielded 1.3 and 0.98 mg
equivalents for Lew and Low Photo respectiveley. All of the
other extraction procedures, except that of one cultivar in
extraction procedure three, failed to yield significant
amounts of zeatin. The possibility exists that perhaps
Table 9s Peak areas and jug equivalents of zeatin for each sample eluant subjected to HPLC.
Sample eluant Peak area fig equivalent equivalent of (cm2) of zeatin zeatin in sample
(*» g)
Zeatin 0.70 5.5 5.5
Lew El H2O/NH4OH 0.62 4.9 0.0 Low Photo El H2O/NH4OH 0.54 4.2 0.0
Lew E2 original 0.57 4.5 0.0 HoO/EtOH. 0.64 5.0 0.0
Low Photo E2 original -— -—
B20/EtOH 0.62 4.9 0.0
Lew E3 H2O 0.00 3.9 0.0 Low Photo E3 H2O 2.34 18.3 171
Lew E4 original 2.40 18.8 355 H 2° 1.69 13.2 205 NELOH 1.24 9.7 112
Low Photo E4 original 1.49 11.7 165 H2° 1.07 8.4 77 NB4OH 1.89 14.8 248
^Equivalent amount of zeatin present in this sample was not computable due to the erratic nature of the chromatogram.
67
extraction procedures one and two could be made to work if
precautions against oxidases and other enzymic activity (as
well as working at low temperatures) could be taken. It
seems unlikely, however, in view of the fact that these two
extraction procedures make use of extremely simple solvent
systems. The solvent system used in extraction procedures
three and four is more suitable to the extraction of
cytokinins based on the mixture of compounds of varying
polarity and the fact that more types of cytokinins will be
soluble in it. It also prevents oxidase and phosphatase
activities due to the presence of a free radical trap which
removes or binds reactive compounds such as enzymes. It
seems likely that with this solvent, a suitable extraction
procedure could be worked out which utilizes less tissue.
The results presented indicate that 100 g are sufficient for
the extraction of zeatin in detectable amounts. It is
difficult to work with 100 gf however, and it would be ideal
if detectable amounts of zeatin could be extracted from less
tissue. Extraction procedure three looks promising since
342 /XG of zeatin were obtained from 10 g of Low Photo
leaves. Perhaps with a better functioning column system and
a little more finesse, this procedure could be made to work
repetitively.
Differences between cultivars within extraction
procedures also exist. They do not appear to be significant
with extraction procedures one and two. With extraction
68
procedure three, Low Photo yielded 5 times more zeatin than
did Lew, whereas with extraction procedure four, both
cultivars yielded about the same amount. Nothing certain
can be stated about these differences until the extraction
procedures are rendered repeatable. In other words, it
cannot be stated safely that significant differences between
cultivars in their ability to produce zeatin exist. It will
be possible, however, to detect such differences once the
column system of the extraction procedure has been
optimized.
Shin Lay&L chromatography
The Rf values for all of the extracts subjected to
thin layer chromatography are summarized in table 10. The
numbers listed are mean values for three repetitions of each
eluant. Zeatin has an Rf value of 0.80 in this system, and
all spots within +/- 0.02 Rf units of 0.80 are considered to
be zeatin. The intensity of fluorescence increased with the
amount of tissue used in the extraction. Perhaps for this
reason no spots were readily detectable for either cultivar
subjected to extraction procedure one. The H20 washes for
both cultivars subjected to extraction procedure two did
show a spot which eluted at the same rate as zeatin. The
spots were extremely faint and barely detectable which would
indicate that very little zeatin is present. The fact that
the HPLC did not pick up any detectable amounts of zeatin
69
Table 10: Rf* values for eluants subjected to TLC.
Extract Eluant Rf values
Lew El Low Photo El
Lew E2
Low Photo E2
Lew E3
Low Photo E3
Lew E4
Low Photo E4
H2O/NH4OH H2O/NH4OH
original H20
original H2O
HOO
Ho0
original H2O
NH4OH
original H20
NH4OH
0.83 0.97 0.82 0.85 0.82
0.98 0.87 0.83 0.97 0.85 0.82
0.44 0.81 0.73 0 .82 0.58 0.44 0.79 0.67 0.79 0.52
Rf values are a measure of the distance of a spot from the start relative to the distance of the Bolvent front from the 8tart.
70
from this extraction procedure indicates that the amount of
zeatin present in these extracts is not enough to overcome
the variability inherent with the system. In other words,
the amount of zeatin present could easily not be considered
significantly different from the amount of zeatin each
sample was spiked with if it does not increase the area of
the peak beyond the range of values calculated based on the
error involved with spiking.
The H2O wash of the Low Photo cultivar subjected to
extraction procedure three showed the elution of a spot at
the same rate as that for zeatin. The chromatogram for the
water wash of the Lew cultivar did not indicate the presence
of any such spot. There is a spot, however, which eluted at
a slightly faster rate than zeatin which could be one of its
derivatized or break down forms. Perhaps this is the reason
that a significant amount of zeatin was not detected in this
cultivar on the HPLC.
The chromatograms for the H20 and NH4OH eluants of
both cultivars subjected to extraction procedure four showed
the presence of spots which eluted at the same rate as
zeatin. The original eluants for both cultivars were very
concentrated and as a result the separation was poor.
Instead, a very slow migrating spot was detected which was
extremely concentrated by all of the compounds present in
the extract. Better separation was not obtained by diluting
the extract, so the problem evidently lies in the system.
71
Perhaps a different solvent system or a different solid
phase would enhance the separation. Without pressurer a lot
of the separations obtained on the HPLC will not be obtained
by other chromatographic methods.
The only conclusions that can be drawn from the TLC
results is that zeatin could be present in those eluants
whose chromatograms showed spots which migrated at the same
rate as the standard for zeatin. No comparisons between the
results obtained by this method and the results obtained on
the HPLC will be attempted since the two systems are so
different.
To further verify that the spots obtained on the
chromatograms are indeed zeatin, they could be scraped from
the plates and subjected to analysis by one of the bioassays
specific for cytokinins. For this study, it was hoped that
a bioassay which was quick and sensitive could be utilized.
Several attempts to effectuate an agreement between this
system and either the barley leaf senescence bioassay or the
radish cotyledon bioassay ended in failure.
Barley Leaf Senescence Bioassay
A brief representation of the type of data obtained
from the barley leaf senescence bioassay is illustrated in
figure 18. The data exhibited are a compilation of eight
experimental runs which distinctly exemplify the discordant
nature of this bioassay. It should be noted that only the
72
attempted standard curves, utilizing kinetin as the active
compound, are presented. Several trials using the TLC
scrapings were attempted, but as suspected, the data were as
inconsistent as those obtained for the standard.
Radish Cotyledon BioafiSay
Figure 19 presents a compilation of data from two
experimental repetitions of the radish cotyledon bioassay
utilizing zeatin as the active compound. The data look
promising and indicate the potential for this bioassay,
however, the problem encountered was one of contamination.
Other experimental runs were attempted with both a standard
and the TLC scrapings, but the data were impracticable due
to contamination.
KINETIN CONC (3.0fig/ml)
18J Attempted standard curve for the barley leaf senescence bioassay, utilizing kinetin as the active compound. The abscissa represents dilutions of a 3.0 /ig/ml stock solution.
0.5-i
0.4-
AVG WT (g r )
0.3-
0.2-
0.1-
00 h ,-5 T6 '-7 10 7 KT 10" 10
ZEATIN CONC. (3.0uglml)
10®
Figure 19: Attempted standard curve for the radish cotyledon bioassay, utilizing zeatin as the active compound. The abscissa represents dilutions of a 3.0 jig/ml stock solution.
si
SUMMARY AND CONCLUSIONS
Leaf tissue from two strains of alfalfa were
subjected to four extraction procedures specific for the
isolation of cytokinins. Identification was restricted to
only one cytokinin, namely zeatin.
The ultraviolet spectra were used as a means for
determining which extracts contained detectable amounts of
zeatin and which did not. Those extracts yielding prominant
spectra were then subjected to analysis by reverse phase
high performance liquid chromatography. They were also
subjected to thin layer chromatography in an attempt to
isolate the zeatin fraction. These fractions were then
tested for biological activity by both the barley leaf
senescence bioassay and the radish cotyledon bioassay.
Differences between the extraction procedures in the
amount of zeatin-like compounds isolated were noted. In
general, it appears that extraction procedure four yielded
more zeatin-like compounds, although extraction procedure
three showed potential. An attempt to explain these
differences was based on the differing amounts of tissue
used in each procedure as well as the presence or absence of
an enzyme inhibitor in the extraction media.
No differences in the amount of zeatin-like
compounds present can safely be stated to occur between the
75
76
two cultivars or strains. The variability involved with
each procedure did not allow such conclusions to be reached,
but a reduction in this variability by optimizing the column
system could permit such differences to be measured.
Compounds with Rf values similar to that for zeatin
were separated by thin layer chromatography. The biological
activity of these compounds could not be measured due to the
failure of either bioassay system to perform. These
separated compounds, in conjunction with the HPLC results,
suggest that zeatin-like compounds have been isolated from
alfalfa leaves.
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