AN ABSTRACT OF THE THESIS OF
LOGAN ALLEN NORRIS
for the
(Name of student)
in Botany (Plant Physiology)
(Major)
presented on
Ph, D.
(Degree)
er-
/5 (96.
(Date)
Title: QUALITATIVE CHANGES IN CYTOPLASMIC PROTEINS IN
PLANTS TREATED WITH PLANT GROWTH REGULATING
CHEMICALS
Abstract approved:
Redacted for Privacy
R. 0. Morris
This thesis concerns the alteration of the course of selective
protein synthesis and morphogenesis in plants treated with growth
regulating chemicals. Treatment of 48-hour-old "Alaska" pea seedlings (Pisum sativum L.) with 2, 4-D, IAA, NAA or picloram caused
inhibition of epicotyl and primary root elongation and proliferation of
massed lateral roots. Squash (Cucurbita maxima L.) and corn (Zea
mays L.) seedlings also produced abnormal lateral roots after 2, 4-D
treatment.
The lateral roots induced by 2, 4-D in pea seedlings were initi-
ated in the pericycle opposite all xylem poles over the length of the
root between 6 and 9 hours after treatment. Lateral roots in control
pea seedlings were also initiated in the pericycle but at a later time
and were restricted to widely separated centers opposite a single
xylem pole at a given point in the root.
The soluble cytoplasmic proteins of plant root sections were
fractionated by anionic gel electrophoresis in 10% acrylamide gels.
Some proteins were common to all sections of pea roots but others
varied quantitatively with position in the root. A few proteins were
found only in certain portions of the root and may be associated with
root development. Proteins with an Rf of 0. 41 (band 0.41) were
prominent members of the latter group. Corn and squash seedlings
gave similar results including the prominent changes in the intensity
of band 0.41. However, the electrophoretic patterns were markedly
different among species.
The electrophoretic patterns of proteins from roots of 2, 4-Dtreated corn and squash seedlings and from roots of 2, 4-D-, IAA-,
NAA- or picloram- treated pea seedlings differed quantitatively and
qualitatively from patterns associated with untreated seedlings. Band
0.41 appeared in all sections and increased in intensity with distance
from the root tip and time after treatment. In pea seedlings, band
0.41 was visible 12 hours after treatment with 2, 4-D. Pea seedlings
preferentially incorporated alanine-C14 into proteins in band 0.41
between 6 and 12 hours after 2, 4-D treatment suggesting that synthesis of these proteins was initiated during this interval. The distribution of band 0.41 and the pattern and timing of lateral root develop-
ment were clearly associated in primary root sections of control,
2, 6-D- and ethylene-treated pea seedlings.
The close temporal and spatial association of band 0.41 with the
initiation of lateral roots suggests a cause and effect relationship
between these events. The identity of the proteins in band 0.41 is
unknown, and there is no assurance band 0.41 from different root
sections and species contains the same proteins. A procedure is
described for the isolation of the proteins in band 0.41 by preparative
scale gel electrophoresis.
Qualitative Changes in Cytoplasmic Proteins in Plants
Treated with Growth Regulating Chemicals
by
Logan Allen Norris
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
June 1970
APPROVED:
Redacted for Privacy
Assistant Professor of Agricultural Chemistry
Redacted for Privacy
Professor of Cflginistry
Redacted for Privacy
Associate Professor of Botany
Redacted for Privacy
Chairman ol/bepartm.ent o
otany and Plant Pathology
Redacted for Privacy
Dean of Graduate School
Date thesis is presented
Typed by Opal Grossnicklaus for
Logan Allen Norris
I lovingly dedicate this thesis
to my wife, Betty.
She suffered the pain of the failures and
relished the joys of the triumphs with me. Her
unselfish support, patience and understanding
made the completion of this degree program
possible.
ACKNOWLEDGMENT
I gratefully acknowledge:
R. 0. Morris for his conscientious and stimulating
supervision of my thesis research;
V. H. Freed for his encouragement and long support
of my education and research;
R. R. Becker, W. K. Ferrell and T. C. Moore for their
unique ability to conduct a well organized and chal-
lenging classroom instructional program along with
an active research program;
and
the Herman Frasch Foundation for financial support of the
thesis research.
TABLE OF CONTENTS
INTRODUCTION
1
REVIEW OF LITERATURE
7
Gene Expression and the Control of Protein Synthesis
Relation of Protein Synthesis to Morphogenesis
Biological Activity of Some Growth Regulating Chemicals
Mode of Action of Some Growth Regulating Chemicals
Relation of Protein Complement to Specific Plant
Structures or Functions
MATERIALS AND METHODS
7
12
15
18
23
28
28
Germination of Plants
Purification and Application of Growth Regulating
29
Chemicals
32
Anatomical Investigations
32
Extraction of Soluble Cytoplasmic Proteins
33
Determination of Protein
Analytical Scale Polyacrylamide Gel Disc Electrophoresis 34
Preparative Scale Polyacrylamide Gel Disc Electrophoresis 39
Incorporation of Radioactive Alanine into Soluble Cyto40
plasmic Proteins
43
Isolation of a Particular Protein
RESULTS
47
47
Morphology of Plants Treated with Regulating Chemicals
47
Pea Seedlings Treated with 2, 4-D
Pea Seedlings Treated with IAA, NAA, or Picloram 67
68
Squash and Corn Seedlings Treated with 2, 4-D
68
Anatomy of Roots of 2, 4-D- Treated Pea Seedlings
Gel
ElectrophorDevelopment of Techniques for Analytical
73
esis
73
Extraction and Electrophoresis
75
Protein Staining
77
Analysis of Electrophoretic Patterns
82
Proteins
Electrophoretic Behavior of Soluble Cytoplasmic
82
Proteins from Control Pea Seedlings
86
Proteins from 2, 4-D- Treated Pea Seedlings
91
Incorporation of Alanine-C" into Proteins
IAA,
Proteins from Pea Seedlings Treated with
104
NAA or Picloram
TABLE OF CONTENTS (CONTINUED)
Proteins from Pea Seedlings Treated with Ethylene
Proteins from Pea Seedlings Treated with 2, 6-D
Proteins from Control and 2, 4-D-treated Squash and
Corn Seedlings
Isolation and Stability of Induced Proteins
DISCUSSION
Morphology of Plants Treated with Growth Regulating
Chemicals
Anatomy of 2, 4-D- Treated Pea Seedlings
Electrophoretic Behavior of Plant Proteins
Incorporation of Radioactive Alanine into Proteins of
Band 0.41
Proteins from Pea Seedlings Treated with IAA, NAA,
or Picloram
Proteins from Pea Seedlings Treated with Ethylene or
2, 6-D
Band 0.41
CONCLUSIONS
109
111
114
117
127
128
129
131
133
134
135
136
141
BIBLIOGRAPHY
143
APPENDIX
153
LIST OF FIGURES
Page
Figure
1.
Pump and sprayer for applying IAA, NAA, or
alanine-C" to pea seedlings.
31
2.
Analytical gel electrophoresis apparatus.
36
3.
Zones of elongation of primary roots of pea seedlings
48 and 72 hours of age.
48.
4.
a, b, c, d, e. Morphogenesis of pea seedlings between
49
48 and 168 hours of age.
4a.
Control seedlings at 48, 60, and 72 hours of age.
49
4a.
(cont'd) Control seedlings at 96 and 168 hours of age.
50
4b.
2, 4- D- treated seedlings at 60 and 72 hours of age.
51
4b.
(cont'd) 2, 4 -D- treated seedlings and a root tip at
96 hours of age.
52
4b.
(cont'd) 2, 4-D-treated seedlings and a root tip at
168 hours of age.
53
4c.
NAA-treated seedlings at 60 and 72 hours of age.
54
4c.
(cont'd) NAA-treated seedlings and a root tip at 96
hours of age.
55
4c.
(cont'd) NAA-treated seedlings and a root tip at
168 hours of age.
56
4d.
IAA-treated seedlings at 72 hours of age and seedlings
and a root tip at 96 hours of age.
57
4d.
( cont'd) IAA-treated seedlings and a root tip at 168
hours of age.
58
4e.
Picloram-treated seedlings at 60 and 72 hours of age.
59
4-
(cont'd) Picloram-treated seedlings and a root tip at
60
96 hours of age.
LIST OF FIGURES (CONTINUED)
Page
Figure
4e.
(cont'd) Picloram-treated seedlings and a root tip
at 168 hours of age.
61
5.
Morphogenesis of squash seedlings between 72 and
196 hours of age.
62
5.
(cont'd) Control and 2,4-D-treated squash seedlings
63
6.
Morphogenesis of corn seedlings between 60 and 180
hours of age.
64
6.
(cont'd) Control and 2, 4 -D- treated corn seedlings at
84, 108, and 180 hours of age.
65
6.
(cont'd) 2, 4-D-treated corn seedlings and a root tip at
180 hours of age.
66
7.
a, b, c, d.
7a.
Section collected 12 hours after 2, 4 -D treatment.
70
7b.
Section collected 24 hours after 2, 4-D treatment.
70
7c.
Section collected 36 hours after 2, 4 -D treatment.
71
7d.
Section collected 48 hours after 2, 4-D treatment.
71
8.
Cross sections of control pea roots collected 15 mm
below the cotyledons at 120 hours of age.
72
9
Influence of amount and origin of proteins on the Rf of
the "skewed" band in 10% acrylamide gels.
76
10.
a, b, c, d. Diagrammatic, photographic, and spectro-
78
at 120 and 196 hours of age.
Cross sections of pea roots collected 10 mm 70
behind the root tips 12, 24, 36, and 48 hours after exposure to 5 x 10-5 M 2, 4-D between 48 and 50 hours of age.
photometric methods of illustrating the distribution of
soluble cytoplasmic proteins in 10% acrylamide gels
after analytical gel electrophoresis.
LIST OF FIGURES (CONTINUED)
Page
10a.
10b.
10c.
10d.
11.
12.
13.
13a.
13b.
Proteins from 1 to 2 mm behind the root tips of 48-hour- 78
old control pea seedlings.
Proteins from 4 to 5 mm behind the root tips of 96hour-old pea seedlings treated with 5 x 10-5 M 2, 4-D
between 48 and 50 hours of age.
79
Proteins from 5 to 7 mm behind the root tips of 120hour-old squash seedlings treated with 5 x 10-5 M
2, 4 -D between 72 and 78 hours of age.
80
Proteins from 5 to 7 mm behind the root tips of 108hour-old corn seedlings treated with 1 x 10-5 M
2, 4-D between 60 and 62 hours of age.
81
Electrophoretic pattern of soluble cytoplasmic proteins
from sections 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to
7, 7 to 9, and 9 to 11 mm behind the root tips of control
pea seedlings 48 hours of age.
83
Electrophoretic pattern of soluble cytoplasmic proteins
from root sections 10 to 20 mm below the cotyledons of
control pea seedlings 72, 96, and 120 hours of age.
85
a, b, c, d. Electrophoretic pattern of soluble cytoplasmic 87
proteins from sections 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to
5, 5 to 7, 7 to 9, and 9 to 11 mm behind the root tips of
pea seedlings treated with 5 x 10-5 M 2, 4 -D between 48
and 50 hours of age.
87
Electrophoretic pattern of proteins 12 hours after
2, 4-D treatment.
Electrophoretic pattern of proteins 24 hours after 2, 4-D 88
treatment.
13c.
Electrophoretic pattern of proteins 36 hours after
2, 4-D treatment.
89
13d.
Electrophoretic pattern of proteins 48 hours after
2, 4-D treatment.
90
LIST OF FIGURES (CONTINUED)
Page
Figure
14.
14
a through i. The distribution of OD500 and C in
10% acrylamide gels following analytical gel- electrophoresis of soluble cytoplasmic proteins from root
sections of control and 2, 4-D-treated pea seedlings
exposed to alanine-C14.
95
14a.
Proteins from sections 3 to 11 mm behind the root
tips of control seedlings 48 hours of age.
95
14b.
Proteins from sections 3 to 11 mm behind the root
tips of control seedlings 54 hours of age.
96
14c.
Proteins from sections 3 to 11 mm behind the root
tips of seedlings 54 hours of age, 6 hours after treatment with 5 x 10-5 M 2, 4-D.
97
14d.
Proteins from sections 3 to 11 behind the root tips of
control seedlings 60 hours of age.
98
14e.
Proteins from sections 10 to 20 mm below the cotyledons 99
of control seedlings 60 hours of age.
14f.
Proteins from sections 3 to 11 mm behind the root tips
of seedlings 60 hours of age, 12 hours after treatment
with 5 x 10-5 M 2, 4-D.
100
14g.
Proteins from sections 3 to 11 mm behind the root tips
of control seedlings 72 hours of age.
101
14h.
Proteins from sections 10 to 20 mm below the
cotyledons of control seedlings 72 hours of age.
102
14i.
Proteins from sections 3 to 11 mm behind the root
tips of seedlings 72 hours of age, 24 hours after treatment with 5 x 10-5 M 2, 4-D.
103
15.
a, b, c, d. Electrophoretic pattern of soluble cytoplasmic 105
proteins from sections 0 to 1, 1 to 2, 2 to 3, 3 to 4,
4 to 5, 5 to 7, 7 to 9, and 9 to 11 mm behind the root
tips of 96 hour old pea seedlings treated with 1 x 10-5
M picloram between 48 and 52 hours of age, 5 x 10-5
M IAA or 1 x 10-5 M NAA between 48 and 96 hours of
age.
LIST OF FIGURES (CONTINUED)
Page
Figure
15a.
Proteins from sections 0 to 1 or 1 to 2 mm behind
the root tips.
105
15b.
Proteins from sections 2 to 3 or 3 to 4 mm behind
the root tips.
106
15c.
Proteins from sections 4 to 5 or 5 to 7 mm behind
the root tips.
107
15d.
Proteins from sections 7 to 9 or 9 to 11 mm behind
the root tips.
108
16.
Morphogenesis of pea seedlings between 48 and 168
hours of age.
110
17.
18.
Electrophoretic pattern of soluble cytoplasmic proteins 112
from section 0 to 10 mm behind the root tips of 96 and
120-hour-old pea seedlings exposed continuously to 50
ppm ethylene from 48 hours of age.
Electrophoretic pattern of soluble cytoplasmic proteins from sections 0 to 10 mm behind the root tips
of 96 hour old pea seedlings exposed to 5 x 10-5 M
113
2, 6 -D between 48 and 50 hours of age.
19.
a and b. Electrophoretic pattern of soluble cytoplasmic 115
proteins from sections 0 to 1, 1 to 2, 2 to 3, 3 to 4,
4 to 5, 5 to 7, 7 to 9, and 9 to 11 mm behind the root
tips of 120-hour-old squash seedlings exposed to 1 x
10-3 M KH 2 PO4 alone (control) or with 5 x 10-5 M
2, 4-D between 72 and 78 hours of age.
19a.
Proteins from root sections of control squash
,115
seedlings.
19b.
Proteins from root sections of 2, 4-D-treated squash
seedlings.
116
LIST OF FIGURES (CONTINUED)
Page
Figure
20.
a and b. Electrophoretic pattern of soluble cytoplasmic proteins from section 0 to 1, 1 to 2, 2 to
118
3, 3 to 4, 4 to 5, 5 to 7, 7 to 9, and 9 to 11 mm
behind the root tips of 108-hour-old corn seedlings
exposed to 1 x 10-3 M KI-I2P0A alone (control) or
with 1 x 10-3 M 2, 4-D betweeri 60 and 62 hours of
age.
20a.
Proteins from root sections of control corn seed-
118
lings.
20b.
Proteins from root sections of 2, 4-D-treated corn
seedlings.
119
21.
Electrophoretic distribution of ammonium sulfate
fractionated soluble cytoplasmic proteins from sections 0 to 20 mm behind the root tips of 96-hour-old
pea seedlings exposed to 1 x 10-3 M KI-12PO4 alone
(control) or with 5 x 10-5 M 2, 4 -D between 48 and
50 hours of age.
123
22.
Elution of OD280 and Lowry protein from the anode
124
23.
lectrophoretic pattern of proteins from fractions
marked by arrows in Figure 22.
125
24.
Diagrammatic, photographic, and spectrophotometric 127
record of the distribution of proteins isolated from
fractions 25 to 35 (Figure 22 and 23) using the stacking and sieving dual gel preparative scale gel electrophoresis apparatus.
end of a sieving-stacker single gel in the preparative
scale gel electrophoresis apparatus.
LIST OF TABLES
Page
Table
1.
Number of dividing cells per section.
69
2.
Correspondence of bands in 7. 5% and 10% acrylamide
gels.
82
3.
4.
Specific activity of soluble cytoplasmic protein containing alanine-C14 from root sections of control
and 2, 4 -D- treated pea seedlings.
Protein precipitation by ammonium sulfate.
93
121
QUALITATIVE CHANGES IN CYTOPLASMIC PROTEINS
IN PLANTS TREATED WITH GROWTH
REGULATING CHEMICALS
INTRODUCTION
This thesis is concerned with plants treated with growth regulating chemicals. The specific hypothesis is: applying growth regu-
lating chemicals to plants results in alteration of the interdependent
course of selective protein biosynthesis and morphogenesis.
This hypothesis is based on three assumptions. (1) Each cell
has a protein complement which is characteristic of a particular
function, stage of development, or environmental condition. (2) An
alteration in expression of genetic information may be observed at
the tissue level by a change in morphology or function. (3) An altera-
tion in expression of genetic information is manifested at the cellular
level by changes in the protein complement.
The scope of these studies is restricted to the chemicals 2, 4dichlorophenoxyacetic acid (2, 4-D), indole-3-acetic acid, (IAA),
a -napthaleneacetic acid (NAA), 4-amino-3, 5, 6- trichloropicolinic
acid (picloram), ethylene and 2, 6-dichlorophenoxyacetic acid (2, 6-D).
Treatments were applied to peas (Pisum sativum L. , var. Alaska),
corn (Zea mays L., var. Tendermost), and squash (Cucurbita maxima L., var. warted Hubbard). Morphological observations were
made on whole plants; anatomical examinations involved roots of
2
pea plants. Biochemical observations concerned soluble cytoplasmic
proteins from roots of pea, corn, and squash seedlings.
My objectives were to: (1) investigate changes in the protein
complement which accompany morphogenesis induced by growth regu-
lating chemicals, and (2) determine whether changes in the protein
complement were causally related to morphogenesis. These studies
were undertaken to provide some insight into the impact of artificial
growth regulating chemicals on the biochemistry of plants.
Biological differentiation is the acquisition of specialized struc-
tures or functions in the course of development. We do not now understand the mechanisms which control differentiation at the cellular
level.
Differentiation is observed at all levels of organization. The
development of chloroplasts from proplastids after exposure to light
is differentiation at the subcellular level. Spore formation in
Bacillus subtillis is an example of differentiation in a single cell;
Guard cell formation in the epidermis of a leaf is differentiation at
the tissue level. A better understanding of the mechanisms which
control differentiation is important to plant physiologists, to those
concerned with the biological impact of xenobiotics and to those
studying some metabolic disturbances such as cancer in man.
Studies of differentiation in multicellular organisms concern
the questions: (1) How can a single cell, the fertilized egg, give
3
rise to a mature individual of many cell types organized into diverse
tissues each with its own specific functions ? and (2) In the face of
this diversity, how can the basic biochemistry of these cells remain
the same?
The constancy of the basic biochemistry of these cells is understandable based on our knowledge of the genetic control of enzyme
synthesis, the classical chromosome theory of heredity and the
mechanics of somatic cell division. All somatic cells of a given
individual are the same genotype, as illustrated by the phenomenon,
totipotency, i.e., the capacity of a single somatic cell to give rise to
a complete individual. Zeevaart (1965, p. 145) gives an impressive
list of examples of regeneration of complete plants from one or a
few somatic cells. A fundamental question is: How is the genotype
in a totipotent cell modulated to give a particular phenotype?
In the macroscopic world, the phenotype of an organism has
traditionally been considered the result of an interaction between
genotype and environment.
This relationship is true for the expres-
sion of genotype at the cellular level as well. However, the environment of the individual cell is composed of not only the macroscopic
environmental factors but also of certain stimuli such as metabolites,
hormones, or bioelectric potentials from neighboring cells and possibly foreign stimuli like artificial growth regulating chemicals. A
change in the cell environment may result in a change of phenotype
4
of that cell just as a change in the organism's environment may result
in a change in its phenotype.
Differentiation on the cellular level might be defined as the
preferential expression of a portion of the total genome. Haemmerling' s (1963) work with the giant unicellular alga Acetabularia illus-
trates both the residence of the genome in the nucleus and the prefer-
ential expression of part of this genome leading to a distinct morphological event. This alga consists of a rhizoidal part, a stalk (with
the nucleus at the base) and a cap (all part of one cell). The morphology of the cap is species specific. The stalk and cap are regenerat-
ed if they are removed from the base. If the nucleus of one species
of Acetabularia is implanted in the enculeated base of another species,
a stalk and cap are regenerated with the morphology of the species
from which the nucleus was taken. The interaction of cytoplasm
with the implanted nucleus permits expression of that portion of
the genome concerned with regeneration of the stalk and cap (but not
the rhizoid part). However, the particular genotype expressed is
that of the nuclear donor.
An equally impressive example from the laboratory of Gurdon
(1968) illustrates selective expression of genetic information.
A
nucleus from an epithelial cell from the intestine of Xenopus laevis
(frog) was implanted in an enucleated unfertilized egg of the same
species. This pseudofertilized egg was cultured to a completely
5
normal adult frog. This illustrates totipotency of a type, but more
important for our discussion, exposure of the epithelial nucleus to
the environment of the cytoplasm of the egg resulted in a new phenotype.
The one cistron-one polypeptide (one gene- one enzyme) theory
suggests genetic information is expressed through a synthesis of proteins. But most of the genetic information of a given cell is not ex-
pressed at any one time, so most of the genes are not expressed in
synthesis of specific proteins. Control of the availability of genetic
information for protein synthesis is basic to the control of differentiation.
The appearance of new macromolecules such as specific pro-
teins may be regarded as differentiation at the subcellular level.
Such biochemical differentiation may precede cellular differentiation
leading to the formation of new structures or acquisition of new functions. How the production of new proteins ultimately results in new
forms and functions is not fully understood, but logically this relationship between biochemical differentiation and morphogenesis should
exist.
Skoog and Miller (1957) have shown the impact of auxin and
cytokinin growth hormones on differentiation. However, attempts to
link the action of growth regulating chemicals to changes in the ex-
pression of genetic information have been largely unsuccessful.
6
Really definitive experiments have not yet been designed for
three reasons. First, the control mechanisms of protein synthesis
are not completely understood. Increasing evidence suggests these
mechanisms are considerably more complex in higher organisms
than in bacteria. Further, it is difficult to demonstrate an obligatory
relationship between the production of specific macromolecules and
the evolution of specific forms or functions. Finally, most studies of
the impact of growth regulating chemicals have concerned only gen-
eral trends in nucleic acid and protein biosynthesis. The synthesis
of one or more specific macromolecular species associated with
differentiation has not been adequately investigated.
7
REVIEW OF LITERATURE
Gene Expression and the Control of Protein Synthesis
We understand the mechanisms by which the genetic information
of chromosomal DNA is expressed in the formation of specific proteins. Most recent biochemistry texts contain an adequate treatment
of the subject so I need only briefly outline the processes here.
The enzyme RNA polymerase catalyzes the synthesis of RNA
using portions of the DNA molecule as a template to specify the sequence of ribonucleotides. Three general classes of RNA are pro-
duced in this manner, ribosomal RNA (r-RNA), transfer or soluble
RNA (s-RNA), and messenger RNA (m-RNA). The r-RNA and s-RNA
are synthesized over relatively small portions of the DNA molecule,
while m-RNA is synthesized over a much larger portion.
The m-RNA serves as a template for the synthesis of specific
proteins whose amino acid sequences are determined by the ribonucle-
otide sequence of the m-RNA, which in turn reflects the deoxyribonucleotide sequence of the DNA. Protein synthesis occurs on
polyribosomes consisting of several ribosomes on an m-RNA molecule. The ribosomes are made of r-RNA and ribosomal protein.
Amino acids are carried to the site of peptide bond formation by
specific s-RNA species which interact with the m-RNA to insert the
8
amino acid at the correct point giving the m-RNA-dictated amino acid
sequence.
There are only a few species of r-RNA. There are more than
20 s-RNA species since there are one or more distinct s-RNA species
for each amino acid. There are many species of m-RNA. Each pro-
tein or group of proteins must have its own message; so it is clear
why the majority of the DNA codes for m-RNA while relatively small
amount of DNA codes for s- and r-RNA (White, Handler, and Smith,
1964, pages 584-587, 598-610).
Not all the genetic information in the genome of a given cell is
expressed at any one time. The totipotency previously described,
however, clearly illustrates the total genetic complement remains
even in specialized cells. How is the genetic information modulated
so that only selected portions of the genome reach expression in
the synthesis of specific proteins?
Jacob and Monod (1963), in their classical work with bacteria,
proposed that the synthesis of specific proteins is determined by the
rate of synthesis of the m-RNA which codes for that protein. Control
of protein synthesis at this level is called transcriptional control.
According to Jacob and Monod, the genetic material (in bacteria)
consist of structural genes which specify the primary structure of
the protein, regulator genes which determine whether a structural
gene is active or not and an operator gene which is linked to the
9
structural gene. The operator plus one or more structural genes
constitute an operon or transcriptional unit.
The regulator gene produces an allosteric protein called the
repressor which can exist in two conformations. In an inducible sys-
tem, the repressor molecule may block the operator gene preventing
transcription of the structural gene. However, when another chem-
ical (the effector or inducer) such as a hormone, a metabolite, or a
substrate is present the conformation of the repressor changes. It
can no longer block the operator gene, and transcription of the struc-
tural gene can occur. In a repressible system, on the other hand, the
operator gene is not blocked by the repressor alone. Repressor con-
formation changes in the presence of an effector however, permitting
interaction with the operator gene preventing transcription.
This simple and highly specific control mechanism probably
operates in bacteria (Jacob ai d Monod, 1963) and possibly in higher
organisms (Varner, 1964). However, control of protein synthesis
could occur at the translational level as well as the transcriptional
level. Thus, even though a gene might be derepressed and a spe-
cific m-RNA synthesized, the message might not be translated and
the coded protein not synthesized.
There are several points of possible translational control.
For instance, the m-RNA synthesized on the DNA may not be released from the template without interaction with a release factor.
10
Stent (1964) has shown ribosomes are involved in the synthesis and
release of m-RNA from the DNA template. Ribosomes plus a purified preparation of RNA polymerase failed to stimulate in vitro m-RNA
synthesis indicating the need for another material, presumably protein (Shin and Moldave, 1966). Revel and Gros (1967) reported that
a protein factor required for translation of natural m-RNA markedly
stimulated m-RNA synthesis (transcription) in the presence of purified RNA polymerase, ribosomes, and T-4 DNA. Thus, transcription may be under the influence of a protein factor which also modu-
lates translation.
Some specificity of translation is believed to reside in the
s-RNA. Recent advances in column chromatography have revealed
the existence of more than one s-RNA for some amino acids. As
many as six distinct species of s-RNA are reported which accept only
leucine (Anderson and Cherry, 1969).
Soil et al. (1966) and Caskey, Beaudet and Nirenberg (1968)
reported some specificity in codon recognition among s-RNA species
coding for a given amino acid. Vold and Sypherd (1968) observed
changes in the ratios of certain amino acid-accepting species of
s-RNA at different stages of wheat germination. These reports
suggest specific s-RNA species coding for a given amino acid may
be specifically required for synthesis of groups of proteins associated with certain stages of development.
11
Anderson and Cherry (1968) found six leucine accepting species
of s-RNA in the hypocotyl and cotyledons of soybeans. But only four
of these species in the hypocotyl were charged with leucine due to a
lack of tissue specific leucyl-amino-acyl synthetases.
It may appear
there is not sufficient diversity conferred by few s-RNA species or
synthetase enzymes to account for the specificity required for pro-
tein synthesis. However, if entire groups of proteins associated with
particular stages of development are controlled by a specific s-RNAcodon recognition interaction, diversity need not be great. Further-
more, I am confident many more species of both s-RNA and synthetase enzyme will be resolved as the technology of separation improves.
Another potential point of translational control concerns the
availability of the message for translation. Based on patterns of
protein synthesis in sea urchin eggs, Spirin (1966) has postulated
the existence of masked forms of RNA in the cytoplasm. In the
presence of actinomycin-D, which precludes DNA dependent RNA
synthesis (Hurwitz et al., 1962), cleavage to the blastula stage is
attained implicating protein synthesis on preexisting m-RNA. Other
experiments show unfertilized eggs contain m-RNA and the necessary
protein synthesizing apparatus but are incapable of supporting protein
synthesis unless an artificial template or message is provided
(Spirin, 1966).
These reports support the hypothesis that some
m-RNA is not available for translation except under special
12
environmental conditions. The specificity inherent in the transcrip-
tional control hypothesis of Jacob and Monod (1963) also resides in a
masked m-RNA. The interaction of an effector substance such as a
cell metabolite or hormone with a masking agent (probably a protein)
could either be direct or involve an intermediate repressor-like
molecule. This mechanism offers sufficient variability in structure
and conformation to confer a high degree of specificity in the interaction.
The control of macromolecular synthesis in higher organisms
obviously is quite complex. Some control of protein synthesis may
reside at various levels from transcription to the final release of
the polypeptide. But, I stress that whatever the mechanism controlling selective genome expression, the result is the same--synthesis
of a specific complement of proteins.
Relation of Protein Synthesis to Morphogenesis
The presence of specific proteins may lead to differentiation at
the cellular level and interaction among cells may lead to differentiation at the tissue level and ultimately to changes in structure or func-
exists betion. It seems reasonable that an obligatory relationship
tween cellular biochemistry and morphogenesis, but this concept is
difficult to establish experimentally.
Paul and Gilmour (1966) and
Ursprung et al. (1968) have used DNA-RNA hybridization techniques
13
to show organ specific genome translation in rabbit and mouse tissues. The implication is that selectivity in partial genome transla-
tion may lead to specificity of protein synthesis related to organ
structure or function. Some proteins are specifically associated
with a particular tissue or cell type, for instance hemoglobin in erythrocytes, myosin in muscle, and tryosinase in melanocytes. Wheth-
er these proteins are a result of differentiation or a cause of it is
unclear.
The type of experimental data needed to establish an obligatory
relationship between biochemical differentiation and morphogenesis
must include evidence that:
1.
The protein is synthesized before morphogenesis occurs
and
2.
Deletion of the protein prevents morphogenesis.
Some pertinent evidence comes from studies of the development of the slime mold Dictyostelium discoideum by Sussman and
associates(summarized by Sussman, 1966). Dictyostelium spores
germinate into amoebae which feed on bacteria and increase expo-
nentially in number. As the amoebae enter the stationary phase of
growth, they form multicellular aggregates from which a finger-like
projection called the pseudoplasmodium is organized. The pseudo-
plasmodium migrates over the substratum of cells eventually developing a fruiting body on the end of a stalk.
14
Several carbohydrates have been temporally associated with
stages of Detyosteliumdevelopment, but Sussman has concentrated
on an acid mucopolysaccharide containing galactose, galactosamine,
and galacturonic acid. Galactose is incorporated into this polysaccharide by the enzyme UDP-galactose polysaccharide-transferase.
The first enzyme activi ty is detected in early pseudoplasmodia and
the mucopolysaccharide is detected about one hour later.
There are two mutants which do not reach the morphogenetic
stage where the polysaccharide normally appears. They exhibit no
transferase activity and fail to synthesize the polysaccharide.
Two
other mutants develop to the migrating pseudoplasmodium stage,
but develop no mature fruiting bodies. The enzyme appears in these
mutants but is not released or destroyed, events which normally
accompany the final stages of fruiting body development in the wild
type.
These results suggest the enzyme is closely associated with
development of the pseudoplasmodium and fruiting body.
The appearance of the enzyme is actinomycin-D sensitive
up to four hours before it actually appears. Treatment with actinomycin-D before this time suppressed appearance, suggesting de novo
protein synthesis. These results indicate the cistrons coding for the
enzyme are transcribed at least four hours before the message is
translated and may offer additional evidence for the masked forms
of m-RNA Spirin (1966) discussed.
15
These experiments with Dictyostelium satisfy the requirement
that the protein appear before morphogenesis. However, the evidence
is not conclusive that the protein leads to morphogenesis. Failure of
the protein to appear in certain mutants could result from a lack of
morphogenesis, rather than the converse.
The absolute dependence of morphogenesis on the synthesis of
a specific protein could be shown by finding a morphologically deficient organism with a related point deletion. That is, the deletion
of one cistron, and therefore one polypeptide, would result in deviation from normal morphogenesis.
Biological Activity of Some Growth Regulating Chemicals
The growth regulating properties of 2, 4-D, IAA, and NAA have
been recognized for some time. Kiermayer (1963) has reviewed the
effects of these compounds on cell division, elongation, and differentiation. It is sufficient to note that these chemicals are active in the
common bioassays (Avena coleoptile curvature and straight growth
tests, pea stem elongation, and pea split stem tests) for auxin activity. Picloram, 2, 6-D, and ethylene however, need additional con-
sideration.
Picloram is particularly active as an herbicide and performs
consistently as an auxin. Kefford and Caso (1966) reported it was
stimulatory at low concentrations and inhibitory at high concentrations
16
to the growth of wheat and oat coleoptiles, pea stem internodes, and
pea root tips. Picloram was stimulatory at 10-9 M, had no effect at
10-8 M and was inhibitory at 10
-7 M iin the pea root tip growth test.
Picloram also promoted extension of bean epicotyls at 5 x 10-7
M and showed maximum activity in the soybean hypocotyl straight
growth test at 10-5 M to 10-4 M (Eisenger, Morre, and Hess, 1966).
Schranck (1968) noted 10-5 M to 104 M picloram stimulated elonga-
tion of oat coleoptiles but markedly repressed geotropic responses.
Based on the classical auxin bioassays, 2, 6 -D has little activity
in comparison with potent auxins like NAA, IAA, and 2, 4 -D, but bio-
logical activity is relative. Bourke, Butts, and Fang (1962) reported
2, 6 -D inhibited the uptake of glucose by pea root tips as effectively
as 2, 4 -D.
Chkanikov, Kostina, and Gertsuskii (1966) indicated
2, 4 -D and 2, 6 -D in the range of 10-2 M to 105 M behaved exactly
the same as inhibitors of cyclic photophosphorylation in beans.
Finally, Osborne et al. (1955) showed 2, 6 -D was active in the Went
pea curvature test, although 2, 4 -D was considerably more active.
Although 2, 6 -D certainly is not without biological activity, its
activity as an auxin is in question. All authors claim the use of
highly purified materials but low-level contamination of 2, 6 -D by a
highly active auxin like 2, 4 -D seems likely.
Ethylene has been reported to have a growth modifying effect
on several plants. In addition to its association with fruit ripening
17
and leaf abscission, Burg and Burg (1966) reported ethylene inhibited
pea stem elongation but stimulated lateral swelling. They also observed that 1 x 10-6 M IAA stimulated ethylene production in pea stem
sections and suggested inhibition of stem elongation by IAA results
from stimulation of ethylene.
A large number of plants will evolve ethylene in response to
treatment with a variety of growth regulating chemicals. For in-
stance, IAA, NAA, or 2, 4 -D stimulated ethylene evolution from bean,
corn, cotton, and sunflower tissues (Abe les, 1966) and from zebrina,
coleus, tobacco, and coffee plants (Abe les and Rubenstein, 1964).
It may be tempting to suggest that stimulation of ethylene synthesis is the basis for the action of growth regulating chemicals, but
recent research indicates differences in their mechanisms of action.
For instance, ethylene inhibition of excised pea root sections was
influenced by tissue age and solution composition; these factors had
no major effect on IAA inhibition. In addition, conditions which
caused a reduction in IAA-stimulated ethylene evolution failed to
affect the inhibitory action of IAA on root elongation (Andreae et al.,
1968).
Holm and Abe les (1968) felt the herbicidal action of 2,4-D was
not ethylene mediated since plants exposed to 10% ethylene for 48
hours recovered in an ethylene free environment. They reported,
however, that some of the 2,4-D-induced nucleic acid and protein
18
effects might be ethylene mediated since exposure of some plants to
ethylene leads to increases in synthesis of DNA, RNA, and protein.
Holm et al. (1968) reported 2, 4-D and ethylene evoked similar in-
creases in RNA polymerase activity in soybean hypocotyls. These
reports indicate ethylene is biologically active and is evolved from
tissues treated with growth regulating chemicals. The importance of
ethylene in auxin action is in doubt, however.
Mode of Action of Some Growth Regulating Chemicals
Numerous mechanisms of action have been proposed for growth
regulating chemicals. These include alteration of membrane perme-
ability, respiration rate, substrate distribution patterns and many
others. These aspects of auxin action are reviewed by Van Overbeek
(1966) and Galston and Davies (1969).
Penner and Ashton (1966) cite over 300 references dealing spe-
cifically with effects of the chlorophenoxy herbicides at the cellular
level. They included main chapters dealing with the impact of these
herbicides on plant processes involving carbohydrates, lipids, nitro-
gen metabolism, organic acids, ethylene, alkaloids, steroids, vita-
mins, pigments, mineral nutrition, water relations, endogenous
auxins, nucleic acids, enzymes, respiration, and photosynthesis.
Nearly every aspect of plant growth, development, and metabolism
is influenced by the chlorophenoxy herbicides suggesting that
19
compounds like 2, 4-D either have no central mode of action or they
affect a process basic to many metabolic and developmental activities.
Patterns of growth regulating chemical-induced nucleic acid and
protein synthesis have been intensively investigated in recent years.
Silberger and Skoog (1953) first reported auxin-induced nucleic acid
increases which preceded increases in fresh weight of plants.
In
soybean hypocotyl, the maximum concentration of RNA was found
just prior to initiation of cell proliferation in mature, normally nonmeristematic tissue (Key and Hanson, 1961).
Chrispeels and Hanson (1962) felt that the real effect of 2, 4-D
was exerted in the nucleus through DNA-directed RNA synthesis.
In detached leaves, 2, 4-D maintained incorporation of leucine-C14
into protein while a marked decline occurred in untreated leaves
(Osborne and Hallaway, 1964). IAA and NAA stimulated the incorporation of P-32 and C14 -adenine into RNA of nuclei isolated from
pea and mung seedlings and mature onion bulb scales (Maheshwari,
Guha, and Gupta, 1966).
These reports indicate a general effect of
growth regulating chemicals on metabolic activities involving nucleic
acids and proteins.
Metabolic inhibitors have been used to determine which steps
in the sequence of expression of genetic information are important in
auxin responses. RNA and protein synthesis are reported to be
essential for cell elongation in soybean hypocotyls (Key, 1964) and
20
in pea stem sections (De Hertogh et al., 1965).
Stimulatory concentrations of 2, 4-D enhanced nucleotide-C14
incorporation into RNA (primarily r -RNA) from elongating regions
of excised soybean hypocotyls. Inhibitory concentrations decreased
incorporation and Actinomycin-D inhibited auxin-stimulated synthesis of r-RNA (Key and Shannon, 1964). Pretreatment of pea stem
tissue with actinomycin-D (two hours before auxin treatment) completely inhibited auxin-induced elongation. Pretreatment with auxin
and then actinomycin-D (one hour later) resulted in no inhibition of
elongation until two hours after treatment with inhibitor. The twohour effective life of the actinomycin-D-sensitive material suggests
it may be m-RNA (De Hertogh et al.,
1965).
A similar dependence of auxin- stimulated extension growth on
both RNA and protein synthesis are reported for soybean hypocotyls,
maize mesocotyls, and oat coleoptiles (Coartney, Morrie, and Key,
1967) and artichoke tubers (Nooden, 1968). Several studies showed
auxin-induced increases in nucleic acid and protein levels resulted
from de novo synthesis and not retardation of normal degradative
processes (Osborne and Halloway, 1964; Key, 1964; Roychoudhury
and Sen, 1964; Key and Shannon, 1964; Nooden and Thimann, 1965,
1966; Trewavas, 1968).
Auxin-mediated increases in RNA and protein levels may seem
quite significant in auxin action. However, they may have little
21
specific connection with processes of differentiation (Evans and Ray,
1969).
Would similar results be obtained by varying certain envi-
ronmental parameters like light, moisture, and nutrients to maximize
growth potential? A general increased synthesis of RNA and protein
following application of growth regulating chemicals could be a re-
sponse to an increasing growth rate or metabolic activity. A change
in direction of differentiation may require instead, changes in synthesis of specific RNA and proteins. The majority of studies of effects
of growth regulating chemicals on nucleic acid and protein synthesis
have not demonstrated changes in the complement of specific RNA
or protein species.
If transcriptional control of protein synthesis is operative,
chemical alteration of growth requires synthesis of specific new
species of m-RNA, which cannot be detected directly with the present technology. Differential centrifugation indicated heavy incorpora-
tion of radioactive RNA precursors into the ribosomal fraction of
soybean hypocotyls following treatment with 2, 4-D (Key and Shannon,
1964; Key and Lin, 1966). This distribution of label could result
from incorporation of labeled r-RNA into polyribosomes or to a les-
ser extent, incorporation of labeled m-RNA and s-RNA into the protein synthesizing unit, or from some combination of the three.
Chromatography of RNA on columns of methylated albumin on
kieselguhr (MAK) gives neither satisfactory resolution of RNA species
22
nor satisfactory nucleic acid recovery (Masuda and Tanimoto, 1967;
Vanderholf and Key, 1968). Much of the research using MAK column
chromatography needs critical evaluation.
Trewavas (1968) reported IAA-treated pea stem sections prefer-
entially incorporated radioactive precursors into a species of RNA
with a slow rate of turnover (probably r-RNA).
Hybridization exper-
iments between pea DNA and the labeled RNA from these plants also
indicates synthesis of r-RNA with little incorporation of radioactivity
into m-RNA (Trewavas, 19 6 8 ).
Inhibition of auxin-induced growth by actinomycin-D is usually
considered to indicate an obligatory requirement for RNA synthesis
in the growth response. Roychoudhury, Datta and Sen (1965) reported
actinomycin-D inhibited auxin-induced DNA-dependent RNA synthesis
in isolated coconut milk nuclei. However, raising the concentration
of IAA from 10-5 M to 10-2 M partially overcame the inhibitory ef-
fect of the actinomycin-D. It appears that the IAA and actinomycin-D
were acting competitively, perhaps not directly on DNA-dependent
RNA synthesis. Similar results were reported by Datta and Sen (1965)
when they investigated auxin-induced incorporation of amino acids
into protein in the same system. Cleland (1965) found oat coleptile
wall extensibility was not dependent on RNA synthesis, because nor-
mal extensibility was observed in the presence of auxin and sufficient
actinomycin-D to cause 90% reduction in RNA synthesis.
23
Evans and Ray (1969) found extension growth of oat coleoptiles
commenced 10 minutes after exposure to IAA. This response was
actinomycin-D insensitive, and they felt it was too rapid to permit
transcription, translation, and protein action.
Trewavas (1968) reported that cell expansion in auxin-treated
pea stem sections is maximal at the time RNA and protein changes
are just beginning. These reports do not support the hypothesis
that protein and nucleic acid synthesis are obligate prerequisites of
tissue expansion.
Although all investigators do not agree on the requirement for
DNA-dependent RNA synthesis in auxin action, there is general
agreement that protein synthesis is necessary (Datta and Sen, 1965;
De Hertogh et al., 1965; Key, 1964; Masuda and Tanimoto, 1967;
Nooden and Thimann, 1965; Cleland, 1965).
Treatment with inhibi-
tors of protein synthesis has invariably resulted in inhibition of
auxin effects. Auxin-induced growth would seem to require not
protein in general but a particular complement of proteins, that is,
specific proteins which are not present in sufficient quantity or activity in the absence of auxin to result in the growth response.
Relation of Protein Complement to Specific Plant
Structures or Functions
There are reports of specific proteins associated with certain
24
plant tissues, or stages of development. Steward, Lyndon, and
Barber (1965) studied the electrophoretic properties of certain soluble proteins of pea seedlings in polyacrylamide gels. They found
that (1) many proteins were common to all tissues and all stages of
growth; (2) some proteins were tissue specific; and (3) a few proteins
in a given tissue were found only at certain stages of development.
Their results are an illustration of differential expression of genetic
information as preferential synthesis of certain proteins.
Mills and Crowden (1967), with acrylamide gel electrophoresis,
examined the protein complement of developing pea seedlings.
They
reported both qualitative and quantitative variation in electrophoretic
patterns among organs at different stages of development but pointed
out the danger in assuming bands with similar Rf from different sam-
ples are the same protein. Examination of peroxidase, esterase,
and amylase activity in the gels indicated some bands were composed
of more than one protein component.
Sahulka (1967) found only minor electrophoretic differences in
soluble proteins from various root sections of Vicia faba. However,
the general pattern of protein distribution resembled the electrophoretic pattern obtained by Morris (1966) in developing pea seedling
roots. Morris (1966) found at least five bands of protein specifically
associated with certain stages of root development in 48-hour-old pea
seedlings.
25
McCown, Beck, and Hall (1968) examined the protein comple.-
ment of leaves and stems from three different Dianthus species by
electrophoresis in polyacrylamide gels. Most of the highly reproducible bands of protein appeared in extracts from all three species and
many were common to both leaves and stems. A few bands were
characteristic of the variety or the tissue.
Other reports have dealt with the influence of growth regulating
chemicals on protein complement in plant tissues. Sacher, Hatch,
and Glasziou (1963) and Glasziou and Waldron (1964) report IAA
stimulation of an invertase in sugar cane stalks. Inhibition by chloramphenicol suggested de novo protein synthesis. Venis (1964) stud-
ied the enzyme-catalyzed formation of benzoyl-aspartate in pea stem
segments. This reaction was stimulated by treatment with IAA but
was inhibited in the presence of actinomycin-D and puromycin suggesting both DNA-dependent RNA synthesis and RNA-dependent protein
synthesis were involved.
Sudi (1966) reported that IAA, NAA, and 2, 4 -D induced an en-
zyme catalyzing the formation of IAA- or NAA-aspartic acid complexes. Datta Sen, and Datta (1965) observed a 100% increase in the spe-
cific activity of isocitrate lyase in potato tubers treated with 10-5
IAA.
M
Inhibition by puromycin and actinomycin-D suggested de novo
synthesis of RNA and protein were required.
The disappearance of at least one protein from extracts of IAA-
26
treated bean hypocotyledonary hooks has been reported (Spelsberg
and Sarkissian, 1966, Sarkissian and Spelsberg, 1967a; Sarkissian
and Spelsberg, 1967b). Sarkissian and Spelsberg (1967a) also found
treatment with IAA at either 10-3 M or 10-6 M resulted in the ap-
pearance of three new bands of protein after electrophoresis in acrylamide gels. Morris (1966) reported a protein component of pea root
sections which was associated with meristematic zones in control
roots but appeared in all portions of the roots of seedlings 24 hours
after 2, 4 -D treatment.
Patterson and Trewavas (1967) incubated sub-apical sections
of etiolated peas with C14- or H3-labeled amino acids. After expos3-labeled sections to
ing the C14 labeled sections to IAA and the H
buffer, they were recombined, extracted, and analyzed as one sample. The extract was fractionated by differential centrifugation and
column chromatography and fractions were assayed simultaneously
14-H3 ratio implied no change in protein
for C14 and H3. A constant C
complement with treatment. They found an actinomycin -D sensitive
change in the carbon-tritium ratio following treatment with IAA.
Fan and Maclachlan (1967) found a 12- to 16-fold increase in
the specific activity of cellulase in extracts of pea epicotyls three
days after treatment with IAA. Inhibition of the IAA-induced component by puromycin and actinomycin-D implicated de novo RNA and
protein synthesis. Datko and Maclachlan (1968) found pectinase
27
induction by IAA in pea epicotyl sections but 1, 3-glucanase and
pectinesterase activity increased only in proportion to the total protein content.
On the basis of these reports, I feel it is reasonable to expect
some relationship between protein complement and specific tissues
or functions. It is equally reasonable to expect changes in the protein
complement of certain tissues treated with growth regulating chem-
icals. What is yet to be demonstrated is a cause and effect relationship between the induction of a specific protein and morphogenesis.
28
MATERIALS AND METHODS
Germination of Plants
I
Dry pea seeds, Pisum sativum L. var. Alaska were soaked
10 minutes in 95% ethanol, rinsed with distilled water, soaked 10
minutes in 10% Clorox, then rinsed three times in distilled water.
The seeds were then soaked in an aerated solution of 0.1 mM MgCl2
and 3 mM CaCl2 for 8 hours, drained and planted in vermiculite
moistened with 1mM KH2PO4. Germination was in the dark at 27°C.
for 40 hours. Dry corn seeds, Zea mays L., var. Tendermost (Beal
Seed Company, Ontario, Oregon) and dry squash seeds, Cucurbita
maxima L., var. warted Hubbard2 were germinated like the peas
except the inhibition medium contained 50 [1M chloramphenicol and
germination proceeded for 52 and 64 hours respectively. The imbibition period started at 10 AM forpeas and squash and 10 PM for corn.
Thus, all subsequent chemical treatments were applied at the same
point in the daily growth cycle.
1
Seeds were a gift from W. Brotherton Seed Co., Moses Lake,
2
Seeds were a gift from Floyd Ashton, Univ. Calif., Davis.
Wash.
29
Purification and Application of Growth Regulating Chemicals
The growth-regulating chemicals were from standard laboratory
stock and had been purified by others according to the following methods.
Indo le-3-acetic acid (IAA) and 1 naphthaleneacetic acid (NAA)
(both Maim Res. Lab., N. Y. ) were recrystallized from ethanol-water.
The 2, 4-dichlorophenoxyacetic acid (2, 4-D)(Mann Res. Lab., N. Y. )
was washed with hot hexane and recrystallized first from benzene
then from ethanol -water . The 4-amino-3, 5, 6 -trichloropicolinic acid
(picloram) (Dow Chemical Co., analytical standard) was recrystal-
lized from ether-benzene.
The 2, 6-dichlorophenoxyacetic acid (2, 6-D) was prepared by
refluxing one mole 2, 6-dichlorophenol (Aldrich, D7020) with one mole
monochloracetic acid (Eastman 68) in 150 ml water (pH 9) at 100°C
for 2 hours. The mixture was adjusted to pH 6 with HC1 and steam
distilled to remove unreacted phenol. On cooling, the residue was
acidified to pH 1.
The precipitate was washed with cold water and
then hot hexane. The 2, 6-D was recrystallized first from benzene
and then from ethanol-water.
For treatment, seedlings were rinsed and placed in aerated
solutions of 1 mM KH 2 PO4 containing picloram,
2, 4-D or 2, 6-D
at the appropriate concentration. After exposure they were rinsed
30
three times with distilled water and replanted in vermiculite.
It was necessary to expose seedlings continuous ly to IAA or NAA
to obtain the desired growth response. For this exposure, seedlings
were placed, roots down, on a coarse mesh stainless steel screen.
The screen was supported by a plexiglass frame set in a plastic pan
which served as a sump. The treatment solution (1mM KH 2 PO4 containing IAA or NAA) was pumped to a chromatogram sprayer con-
nected to an air line. The liquid and air flows were adjusted to give
a spray of about 5 seconds duration every 10 seconds (Figure 1).
The treatment solution was completely changed every 12 hours. At
the end of the treatment period the seedlings were rinsed three times
with distilled water and replanted in vermiculite.
For exposure to ethylene (0. 1% ethylene in N2 from Matheson,
Newark, Calif.), seedlings were suspended, roots down, on coarse
mesh stainless steel screen in a 3-liter glass chamber. A small
petri dish containing 5 M NaOH was placed in the bottom to absorb
respired CO2. Seedlings were exposed to 50 ppm ethylene in air at
50 ml/min in the dark at 27°C. for 48 hours. Control plants were
handled in exactly the same manner as treated plants except the
treatment solution or gas lacked the growth regulating chemical.
Pea seedlings (48-hours-old) were treated with 5 x 10-5
M
2, 4-D for 4 hours, rinsed three times with distilled water and the
roots lightly blotted with tissue. The apical 10 mm of the roots were
31
Figure 1. Pump and sprayer for applying IAA,
NAA, or alanine-C14 to pea seedlings.
32
marked at 1 mm intervals with India ink under a dissecting microscope
The seedlings were replanted in vermiculite and at intervals
the position of the India ink marks were determined on 15 seedlings.
The experiment was repeated with control pea seedlings.
Anatomical Investigations
Roots 15 mm long were killed and fixed in FAA (Johansen, 1940),
dehydrated in a tertiary butyl alcohol series and infiltrated with parawax according to Johansen (1940). Groups of five roots were embed-
ded in Paraplast (melting point 56-57°C).
Fifty 10 micron serial sections were taken 5 mm and 10 mm
behind the root tip with a rotary microtome. Sections were stained
with safranin and fast green in clove oil (Johansen, 1940, pages 59,
62) and permanently mounted in Harleco Synthetic Resin. Ten roots
were sectioned for any given treatment or sampling time.
Extraction of Soluble Cytoplasmic Proteins
All operations were conducted in ice in a cold room (2-4°C).
Tissue samples were collected with new razor blades. If serial sec-
tions were taken, several new blades were bolted together with spacers between each blade to give the desired section size. The tip of
the root was placed at right angles to the blades and sectioned most
of the way through by gently pressing with a dry microscope slide.
33
The cuts were completed by rolling a glass rod covered with surgical
tubing across the roots, and the desired sections were placed directly
into the homogenizer. No more than 50 seedlings were sectioned with
a set of cutting edges.
Tissue samples were gently ground in 1 ml Dual glass homogenizers (Ace Glass Co. ) with homogenization medium (Solution 1, Ap-
pendix) consisting of 0.4 M sucrose, 20 mM Tris-chloride, 3mM
o
CaCl2 and 0.1 mM 1, 4-dithiothreitol (pH 6.9 at 0 C. ). The homoge-
nate was centrifuged at 23,500 x g for 30 minutes (3°C. ). The result-
ing crude supernatant (used in most experiments) was pipetted into a
clean tube and kept in ice.
Gentle mechanical homogenization in a near neutral homogenization medium containing 0. 4 M sucrose and a divalent cation will main-
tain the integrity of most cell organelles (Allfrey,
1964).
1959; De Duve,
Thus, the above methods result in a supernatant of soluble
cytoplasmic proteins with minimum contamination from nuclei,
mitochondria, and ribosomes.
Determination of Protein
Protein concentration of plant extracts was determined by the
method of Lowry et al. (1951). In some cases the protein content of
the crude supernatant was measured without prior trichloroacetic acid
(TCA) precipitation. In these instances, 0.05 ml sample and 0.15 ml
34
homogenization medium were added to 3.0 ml of 2.0% Na2CO3, 4.0%
20 and 0.02% NaK-Tartrate (Solution 4 Appendix). After 10 minutes, 0.3 ml of Folin's phenol reagent
NaOH, 0.01% CuSO4
5H
(solution 5-Appendix) was added with shaking and the mixture allowed
to stand for 30 minutes at room temperature. The optical density at
750 nm was determined with reference to a homogenization medium
reagent blank.
A more accurate measurement of protein content was obtained
with a TCA precipitation step.
An aliquot of sample (less than 0.5
ml) was added to 3.0 ml 50% TCA. After 30 minutes in ice, the
sample was centrifuged at 12,000 x g for 10 minutes. The supernatant was carefully decanted and a measured volume of 1.0 M NaOH
added.
This material was allowed to stand overnight at room temper-
ature. The protein content of the NaOH- soluble fraction was measured
as previously described.
The amount of protein in each sample was determined from a
bovine serum albumin standard curve which was linear between 10
and 150 micrograms protein per determination. Protein measurements without the TCA precipitation step gave values about 30%
higher.
Analytical Scale Polyacrylamide Gel Disc Electrophoresis
The principal analytical tool used in these studies was disc gel
35
electrophoresis based on methods of Ornstein (1964) and Davis (1964).
The gel matrix was composed of polymers of acrylamide (Eastman
5521) cross-linked with N, N'-Methylenebisacrylamide (Bis) (Eastman
8383).
The acrylamide and Bis were recrystallized from chloroform
and acetone, respectively, and stored in vacuo in the dark at 0°C.
Ammonium persulfate and N, N, N', N' -Tetra.methylethylene-diamine
(TEMED) (Eastman 8178) comprised the catalyst system for the poly-
merization reaction. The stock solutions (listed in the appendix)
were reported by Davis (1964) to have a useful shelf life of several
months when stored in the dark in a refrigerator.
The electrophoresis apparatus was constructed of plexiglass by
the Oregon State University Physics Shop. The apparatus (Figure 2)
is 36 cm long, 9-1/2 cm wide, and 14 cm high.
The upper portion
of the apparatus fits into the 700 ml capacity lower reservoir which
contains the anode. The upper portion of the apparatus contains the
cathode in a 1-liter reservoir mounted on top of a box through which
cooling liquid can be circulated. The tubes in which the gels are
poured are open to the upper reservoir, pass through the cooling
chamber and protrude from the bottom. Thus the lower end of the
tube is in contact with the lower reservoir buffer.
This electrophoresis apparatus contains 30 tubes 11 cm long
and 5 mm inside diameter. A series of marks scribed on the tubes
aid in preparing gels of uniform length and to insure uniform
36
Figure 20 Analytical gel electrophoresis
apparatus.
37
migration of the buffer front.
After the apparatus was thoroughly clean and dry, the bottom
end of each tube was sealed with a double layer of parafilm. The
analytical gel solution (Appendix) was degas sed in vacuo for 1 minute
then poured. It was then carefully overlaid with a 1:8 dilution of lower
reservoir stock solution (solution 12, Appendix) to obtain a smooth
flat upper surface when the gel polymerized. This step was absolutely
necessary for adequate resolution. After 30 minutes at room temper-
ature, a line at the top of the gel indicated polymerization was complete and the overlying solution was carefully withdrawn.
The stacking gel solution was degassed and poured on top of the
analytical gel. It was overlaid with a 1:8 dilution of upper gel buffer
stock solution (solution 9, Appendix) to produce a flat upper surface.
Polymerization was complete in about 30 minutes. After rinsing
the upper portion of the tube with upper reservoir buffer and remov-
ing the parafilm, the gels were ready to use.
Samples (up to 0.45 ml) were put on the gels in 0.4 M sucrose
and further stabilized against convection by carefully adding small
amounts of dry P-10 polyacrylamide powder (Biogel P-10, 50 to
100 mesh, Bio-Rad Laboratories). The P-10 was carefully stirred
with a glass rod until a thin gel resulted. This operation was critical
to successful gel electrophoresis. The proper consistency of the
P-10 stabilized sample is difficult to describe and is best learned
38
by experience.
After sample stabilization, the lower ends of the tubes were
immersed in lower reservoir buffer and upper reservoir buffer containing tracking dye carefully added to the upper reservoir.
The electrodes were connected to a Spinco Con-stat constant
current power supply and electrophoresis at 3 ma/tube continued at
24
oC
until the tracking dye migrated 55 mm in the 65 mm analytical
gel. Each tube was sealed when the dye reached the desired point.
When all samples had migrated 55 mm, the power supply was
disconnected and the gels carefully removed by rimming the lower
end of the tube with a water-filled hypodermic syringe. Gels were
stained in 0. 5% amido black in 10% acetic acid for a minimum of 1
hour, then destained in warm 10% acetic acid for 6 to 12 hours to
remove unbound dye. Gels were stored in 10% acetic acid.
For long-term storage gels should be stored in the dark in the
cold. Storage at room temperature in the light results in reduced
stain intensity over a period of months. Narayan, Narayan, and
Kummerow (1966) indicate, however, the degree of fading among bands
is proportional to the original intensity of staining in a given gel.
The gels were examined by one or more of several methods.
One method involved scanning the gel with a Gilford 2410 linear trans-
porter connected to a Gilford 2000 spectrophotometer mounted on a
Beckman DU monochromator. In a few instances, photographs were
39
made of gels against a diffuse light source. The gels were in Klett
colorimeter tubes which provided considerable lateral magnification.
Preparative Scale Polyacrylamide Gel Disc Electrophoresis
The resolving power of acrylamide gel electrophoresis was
used to isolate a particular soluble cytoplasmic protein from a mixture of about 20 prominent components and an unknown number of
minor components.
Preparative and analytical scale gel electrophoresis are based
on the same principles but differ in that the latter only provide separation of proteins in a gel cylinder, while isolation of protein compo-
nents are obtained with the preparative scale instrument by continuing electrophoresis until proteins migrate off the anode end of the gel.
Two gel systems were used in Canalco Corporation's Model PB-1
preparative instrument. One system has a single gel called a sievingstacker single gel and was used for preliminary fractionation. Final
purification was accomplished with a dual gel system composed of
a stacking gel on the top and a separate sieving gel on the bottom.
The latter system is called the stacking and sieving dual gel system.
It has gel properties approaching those of the analytical gel system.
The stock and working solutions for these gel systems are described
in the Appendix.
The bottom of the PD-2/230 column was covered with parafilm
40
before 10 ml of degassed 10% sieving-stacker single gel solution was
poured. It was carefully overlaid with 3 ml of hi() dilated gel buffer
stock solution (solution 19, Appendix). After polymerization (20 min. )
the upper part of the column was rinsed, the parafilm removed, and
the gel was ready to use.
The PD-2/70 column was used for the sieving and stacking dual
gel system. The bottom of the column was covered with parafilm
before 2 ml of degassed 15% sieving gel solution was poured. It was
overlaid with 1:8 dilution of dual gel elution buffer stock solution
(solution 23, Appendix). After polymerization was completed (30
min. ), the overlying solution was removed and 2 ml of degassed 3.5
percent stocking gel solution poured. It was overlain with 1:8 dilution of upper gel buffer stock solution (solution 22, Appendix). After
polymerization (20 min.) the upper part of the column was rinsed, the
parafilm removed, and the gels were ready to use.
Incorporation of Radioactive Alanine Into
Soluble Cytoplasmic Proteins
Dry pea seeds were germinated as previously described. At 41
hours of age 225 seedlings were placed, roots down, in the same
apparatus used for spray treating with IAA and NAA (Figure 1). For
the next 7 hours, these peas were sprayed with 50 µc alanine-U-C 14
(Nuclear Chicago, specific activity 162 me /mm) in 50 ml of 10-4 M
41
KH PO4 and 50 f.LM chloramphenicol.
2
The entire apparatus was en-
closed in a large plastic bag in a fume hood.
After a 7-hour spray period the seedlings were lightly rinsed
with distilled water and divided into one group of 75 seedlings which
-3
was treated with 5 x 10-5 M 2,4-D in 10 M KH2 PO4 for 2 hours
and a control group of 150 seedlings which was treated with 10-3
M
KH2PO4 for 2 hours. At the end of this treatment period the seed-
lings were rinsed and returned to the spray treatment apparatus.
Spraying with radioactive alanine continued for the duration of
the experiment. Samples of the treatment solution were taken at the
start of spraying, 12 hours later, and again at the end of the experiment to determine the pattern of C14 uptake.
Radioactivity was measured with a liquid scintillation counter
(Packard Tricarb 314-EX). Samples were counted in a scintillation
solution of Triton-toluene-Omnifluor (Appendix) which has excellent
counting characteristics for aqueous samples. Samples of control
and 2, 4 -D treated roots were collected and the crude supernatant
prepared as previously described. Aliquots were subjected to an-
alytical electrophoresis (in triplicate) in the 10% acrylamide gel
matrix.
The gels were stained with amido black, destained and scanned
at 500 nm using a Gilford 2410 linear transporter on a Gilford 2000
spectrophotometer. Gels were then sliced laterally into 1.6 mm
42
sections.
Slices from one gel for each treatment were counted individually. The two remaining gels for each treatment were also sliced
but only sections 11 through 20 were counted individually. Sections
1 (origin) through 10 were combined with sections 21 to the buffer
front (pooled samples).
The individual slices were placed in separate scintillation vials
and allowed to air-dry for 6 hours before 0.4 ml of 30% H202 was
added. Solubilization of the slice was usually accomplished in 8 to
12 hours in tightly capped vials in a 50°C water bath. When the sam-
ple was cool, 0.5 ml of distilled water was added and then 15 ml of
scintillation solution. Solubilization of pooled samples required 24
hours in 7.0 ml of 30% H2 0 2" Sample volume was adjusted to 15.0
ml with distilled water and a 1.0 ml aliquot removed for counting.
All the solubilized slices from one gel were spiked with toluene-C
14
(4630 DPM), the samples recounted, and the counting efficiency calculated. The efficiency varied between 57.5 and 59. 6% but was not
a function of position in the gel.
This method of gel slice solubilization yielded 85 to 100% C14
recovery. The incubation temperature should be carefully controlled
around 50 °C; since C1402 can be lost at incubation temperatures of
55 and 60°C (Tishler and Epstein, 1968).
43
Isolation of a Particular Protein
Pea seedlings (48 hours old) were treated for 2 hours with 5 x
10-5M 2, 4 -D in the usual manner.
The lower 15 mm of the roots were
excised 48 hours later and extracted with homogenization medium.
The homogenate was centrifuged at 23, 500 x g for 30 min. (3°C. )
and protein content adjusted to 3 mg Lowry protein per ml.
Ammonium sulfate crystals were dissolved in the supernatant
to 50% of saturation and the solution allowed to stand in ice for 10
min. before centrifugation at 12, 000 x g for 10 min. Additional
(NH
)
4 2
SO
4
to 90% of saturation was dissolved in the supernatant and
after 10 min. in ice, the solution was centrifuged at 12, 000 x g for
10 min.
The pellet was suspended in a minimum volume of dialysis
medium (solution 2, Appendix) and dialyzed for 2 hours against two
changes of dialysis medium. Rapid dialysis was accomplished
by placing the dialysis bag between two arms of a rotating (30 RPM)
glass frame. Rotation completely mixed solutions in and outside the
bag maintaining the most favorable concentration gradient for rapid
dialysis. After dialysis and centrifugation (23, 500 x g for 15 min. )
the supernatant was frozen at -70°C.
Samples of this (NH4)2SO4 purified material were thawed in
ice and carefully layered on top of a 10% sieving-stacker single gel
system in the PD-2/320 column in the preparative scale
44
electrophoresis apparatus. The sample was stabilized against convection with Bio-Gel P- 10 and upper reservoir buffer layered over
the sample. The electrodes were connected to the Spinco constant
current power supply, and protein stacking proceeded at 15 ma
(about 20 minutes). The tracking dye required about 60 minutes to
move through the sieving-stacker single gel at 7.5 ma. The current
was reduced to 2 ma as the tracking dye approached the bottom 5 mm
of the gel.
Elution buffer was pumped across the bottom of the sieving-
stacker single gel at 2.5 ml/min. with a Buchler peristaltic pump.
Absorbance (280 nm) of the Heluate" was monitored with a Vanguard
model 1056 U. V. analyzer, and 30 drop fractions were collected
with a LKB Ultrarack 7000 fraction collector. Appropriate fractions
were combined into dialysis tubing, concentrated in the cold by dehydration with powdered polyethylene glycol 2000 and dialyzed against
homogenized medium (solution 1, Appendix). Small aliquots were
examined by analytical gel electrophoresis and the remainder stored
at -70°C.
Final fractionation was done in a stacking and sieving dual gel
electrophoresis system whose gel characteristics approached those
of the analytical system. A sieving gel with a relatively high concentration of acrylamide was overlain by a stacking gel which was low
in acrylamide and had no sieving properties.
Three attempts were
45
made to achieve separation. In each case the sample, a selected
fraction containing six to eight components from the sieving-stacker
single gel system, was stabilized over the stacking gel with Bio-Gel
P-10 and overlaid with upper reservoir buffer. The first two attempts
used sieving gels 25 cm long which were high in acrylamide (20 and
15%).
Diffuse bands and slow protein elution resulted in fractions
too dilute for further use.
In the third and successful attempt, a 1 cm 15% sieving gel and
a 1 cm 3. 5% stacking gel were poured in the PD-2/70 column. Elec-
trophoresis proceeded at 7 ma through the sample, at 10 ma for
protein stacking in the stacking gel and at 12 ma for dye front migration through the sieving gel. The elution buffer was pumped across
the lower end of the sieving gel at 1.5 ml/min. and fractions were
collected at 1 minute intervals.
Lowry protein determination of certain fractions indicated
"elution" of a protein between 51 and 57 minutes after migration of
the tracking dye from the end of the gel. The contents of tubes 51
through 57 (11 ml) were combined and stabilized with Bio-Gel P-10
above a 1 cm 3.5% stacking gel in the PD-2/70 column. The protein
was concentrated electrophoretically at 15 ma through the stacking
gel (no sieving gel) and into a small dialysis bag fixed over the lower
(anode) end of the column. In about 15 min. the tracking dye migrated
through the sample and stacking gel and entered the dialysis bag.
46
Current was maintained for an additional 5 minutes to ensure that
all the protein had migrated from the stacking gel.
The bag was removed from the end of the stacking gel and the
sample dialyzed for 2 hours at 2 -4 °C against two changes of homog-
enization medium. The volume of the dialysate was 0.55 ml. An
aliquot was subjected to analytical gel electrophoresis for confirmation of purification.
47
RESULTS
Morphology of Plants Treated with
Growth Rejulating Chemicals
The response of seedlings to chemical treatment was easily
separated into immediate effects (up to 48 hours post treatment)
and later effects (between 48 and 120 hours post treatment).
Pea,Seedlings Treated with 2, 4-D
I measured the root growth of control and 2, 4-D-treated seed-
lings to determine which portions of the root showed extension growth.
In control seedlings the first mm behind the root tip showed little
elongation, while zones 1 to 2 mm and 2 to 3 mm showed a fivefold elongation and zones 3 to 4 mm and 4 to 5 mm showed a twofold
or less elongation between 48 and 72 hours of age.
Zones more than
5 mm behind the root tip did not increase in length (Figure 3). Seedlings treated with 2, 4-D showed no primary root elongation.
The morphology of etiolated Alaska pea seedlings during early
development is depicted in Figure 4a. Exposure to 2, 4-D (5 x 10-5
M between 48 and 50 hours of age) caused immediate cessation of
primary root and epicotyl elongation. Radial swelling occurred in
the epicotyl and was particularly pronounced 3 to 5 mm behind the
root tip. Rows of bumps along the root axis 48 hours after
St'
Figure 4a, b, c, d, e. Morphogenesis of pea seedlings between 48 and
168 hours of age. Seedlings were grown in
vermiculite in the dark at 27° C. and were
treated with 1 x 10-3 M KH2 P0a alone (control)
or containing 5 x 10-5 M 2,-4-D- between 48 and
50 hours of age, 5 x 10-5 M IAA or 1 x 10-5 NAA
between 48 and 96 hours of age or 1 x 10-5 picloram between 48 and 52 hours of age., The number
of hours in the figure is total seedling age from
the beginning of the imbibition period.
49
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51
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52
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Figure 4b (cont'd). 2, 4-D-treated seedlings and a root tip
at 168 hours of age.
54
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Figure 4c (cont'). NAA-treated seedlings and a root tip at
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57
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Figure 4d. IAA-treated seedlings at 72 hours of age and
seedlings and a root tip at 96 hours of age.
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Figure 4e. Picloram-treated seedlings at 60 and 72 hours of age.
60
--
.
I
I --t-
96 hours
1111
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PICLORAM
Figure 4e (cont'd). Picloram-treated seedlings and a root
tip at 96 hours of age.
PICLORAM
Figure 4e (cont'd). Picloram-treated seedlings and a root tip at
168 hours of age.
u
um
72 hours
m
o
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2,4 -D
MUM=
Figure 5. Morphogenesis of squash seedlings between 72 and
196 hours of age. Seedlings were grown in verm-
iculite in the dark at 27°C. and were treated with
1 x 10-3 M KH PO 4, alone (control) or with 5 x 10-5
M 2, 4-D between 7G and 78 hours of age. The num-
ber of hours in the figure is total seedling age from
the beginning of the imbibition period.
192 hours
2,4 -0
120 hours
CONTROL
2,4 -D
Figure 5 (cont'd). Control and 2, 4-D-treated squash seedlings
at 120 and 196 hours of age.
60 hours
CONTROL
;Ai
a
k
I 1111
MINIUM
n .111111
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15.4
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Figure 6. Morphogenesis of corn seedlings between 60 and 180
hours of age. Seedlings were grown in vermiculite
in the dark at 27°C and were treated with 1 x
10-3 M KH PO4 alone (control) or containing 1 x 10-3
2
M 2, 4 -D between
ages 60 and 62 years of age. The
number of hours in the figure is total seedling age
from the beginning of the imbibition period.
1
1 -V-
_trt-r
4 4
I
a.....
MNE
$4 hours
SOON
Imam
t
i
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or
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n MMMMM MEMO
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ISO hours
ern
2,4 -0
a ROA SA
111..0
1 MM M MMMM OEM EOM
I
a
U
1
Figure 6 (cont'd). Control and 2, 4-D-treated corn seedlings at 84, 108, and 180 hours
of age.
r- rir
T-T
11111ffil
NENE RV
NUN
inuoNNEE
Isms
Num
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4
- amanita mum
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67
treatment preceded the proliferation of massed lateral roots which
predominated the later effects (Figure 4b).
Pea Seedlings Treated with IAA, NAA, or Piclora.m.
Continuous exposure for 48 hours to a spray of IAA or NAA
(5 x 10-5 M and 1 x 10-5 M, respectively, between 48 and 96 hours
of age) gave a 2, 4-D-like response. NAA caused greater radial
swelling and epicotyl elongation but less intense lateral root proliferation than 2, 4 -D (Figure 4c). IAA gave results similar to NAA ex-
cept that epicotyl and lateral root elongation were more inhibited by
IAA (Figure 4d). Seedlings exposed to IAA or NAA at concentrations
up to 1 x 10-4 M for 2, 4 or 6 hours showed an immediate 2, 4-D-
like response but primary root and epicotyl elongation resumed with-
in 48 hours after treatment.
Pea seedlings treated with picloram (1 x 10-5 M for 4 hours)
showed more pronounced swelling 3 to 5 mm behind the root tips and
greater inhibition of epicotyl elongation than seedlings treated with
2, 4-D (Figure 4e). Figure 4e shows three seedlings which resumed
primary root elongation between 96 and 168 hours of age. About half
of all picloram treated seedlings responded this way and were discarded.
68
Squash and Corn Seedlings Treated with 2, 4-D
In order to compare the effects of growth regulating chemicals
on other species, squash (generally regarded as sensitive) and corn
(resistant) were treated with 2, 4-D. Treatment of Hubbard squash
with 2,4-D (5 x 10-5 M for 6 hours starting at age 72 hours) caused
immediate inhibition of primary root and epicotyl elongation. Pro-
liferation of lateral roots occurred but it was not as extensive as in
peas and was restricted to the area just behind the root tip (Figure 5).
Corn treated with 2, 4 -D (1 x 10-3 M between 60 and 62 hours
of age) showed some effects similar to those of 2, 4-D in peas. Cole-
optile elongation ceased but resumed later at a slow rate. Primary
root elongation was inhibited but some proliferation of lateral roots
did occur (Figure 6).
Anatomy of Roots of 2, 4-D- Treated Pea Seedlings
Cross sections of roots from control and 2, 4-D-treated pea
seedlings were examined to determine the time and location of cell
divisions leading to initiation of lateral roots.
I counted the number of cells per section with prominent nucleoli
or condensed chromatin and assumed these cells would soon divide
or had recently done so (Swanson, 1957. ) (Table 1). Each value
respresents the mean of 25 observations from five sections of five
69
different roots. A t-test (Li, 1957) indicated no significant difference
in the number of cells per section with prominent nucleoli or condensed chromatin from control and 2, 4-D-treated seedlings either
3 or 6 hours after treatment. Differences between control and treated seedlings were significant 9 and 12 hours after treatment however,
indicating cell divisions leading to lateral root proliferation were
initiated between 6 and 9 hours after 2, 4-D treatment. Lateral root
initiation occurred around all xylem poles at all levels of the root (Figure
7).
Cell divisions were absent in the cortex and infrequent in xylem
parenchyma. Thus, 2, 4-D-induced cell division were restricted to
the pericycle and then only opposite the xylem poles.
Table 1. Number of dividing cells per section.
Time after treatment
(hours)
Number of cells with condensed chromatin
or prominent nucleoli/section*
CONTROL
TREATED
3
155
164
6
170
158
9
155
273**
12
188
325**
*Mean of 25 observations for each point (five sections from
each of five roots).
**Difference between control and treated significant at 5% level.
Figure 7a, b, c, d. Cross sections of pea roots collected 10 mm
behind the root tips 12, 24, 36, and 48 hours
after exposure to 5 x 10-5 M 2, 4 -D between
48 and 50 hours of age. Roots were killed
in FAA, sectioned at 10 microns in Paraplast,
and sections stained with safanin and fastgreen.
70
Figure 7a. Section collected 12 hours after 2, 4-D treatment.
Figure 7b. Section collected 24 hours after 2, 4-D treatment.
71
Figure 7c. Section collected 36 hours after 2, 4-D trea
Figure 7d. Section collected 48 hours after 2, 4-D treatment.
72
figure 8. Cross sections of control pea roots collected 15 mm
below the cotyledons at 120 hours of age. Roots were
killed in FAA, sectioned at 10 microns in.Paraplast,
and sections stained with safranin and fast-green.
73
In control seedlings, lateral roots normally emerged in sections 10 to 20 mm below the cotyledons around 120 hours of age.
Cell divisions leading to lateral root initiation were observed at 96
but not at 72 hours of age. These divisions were never found around
more than one xylem pole per section and many sections contained
no centers of cell division (Figure 8).
Development of Techniques for Analytical
Gel Electrophoresis
Extraction and Electrophoresis
The reproducibility and resolution of protein patterns obtained
with Morris' (1966) techniques were not adequate. A fairly intense
background stain and the presence of an intensely stained skewed
band with an Rf which varied between 0.6 and 0.8 were particularly
perplexing.
The interference from the skewed band and the high background
intensity were reduced by increasing the level of sucrose in the
homogenization medium to 0.4 M and by stabilizing samples against
convection over the stacking gel with Bio-Gel P-10. Increasing the
level of acrylamide in the analytical gel from 7. 5% to 10% decreased
Rf values but markedly improved resolution.
I tried several other procedures but they did not improve
resolution or decrease the background. Adding 10% sucrose to the
74
spacer gel to prevent gel deformation or increasing the level of dithiothreitol from 0.1 to 0.2 mM (McCown et al., 1968) did not improve
results. Adding polyvinylpyrolidone (PVP) (Loomis and Battaile,
1966) to remove phenols and quinones from the homogenate did not
significantly change the electrophoretic pattern. Precipitation of
proteins by ammonium sulfate (90% of saturation) followed by resuspension in and dialysis against homogenization medium eliminated
some components with Rf values greater than 0.7 but decreased
clarity of the protein patterns.
The optimum amount of protein to apply to an analytical gel
varies with the diameter of the gel and the number of proteins in
the sample. My preparations from pea roots usually contained about
20 distinct bands over a continuous background of stain which de-
creased with increasing Rf. I found 150 to 350 p.g Lowry protein per
gel was a useful range. I usually examined 150 and 300 µg of protein
from each extract to be sure of the location of minor components
and to identify any interaction between Rf and amount of protein.
To learn more about the skewed band I extracted proteins
from sections 0 to 2 mm, 2 to 4 mm and 4 to 6 mm behind the root
tips of control pea seedlings. Various amounts of these proteins
and of a composite sample containing equal amounts of protein from
the 0 to 2 mm and 4 to 6 mm sections were subjected to analytical
gel electrophoresis. The Rf of the skewed band was related to the
75
amount of protein applied to the gel and also to the portion of the root
from which it was extracted (Figure 9). Morris (1966), Sahulka
(1967) and McCown et al. (1968) all reported the presence of a
skewed band or bands which showed variations in Rf with the amount
of protein applied to acrylamide gels.
Protein Staining
Amido black is a general protein stain (Davis, 1964). The
amount of stain bound is linear up to 10 µg protein per band with a
standard deviation of 4% (Fambrough, Fujimura and Bonner, 1968).
Narayan, Narayan and Kummerow (1966) reported amido black
stained bands were reduced in intensity after several months storage. The loss was proportional to the original stain intensity,
however, and relative intensity among bands changed very little.
Coomassie blue has also been used as a general protein stain
in acrylamide gel electrophoresis (Chrambach et al., 1967; Fazekas
et al., 1963). I found gels stained with coomassie blue and amido
black had the same patterns and relative band intensities. The coomas-
sie blue was twice as sensitive as amido black but the bright blue
background interfered with gel analysis. Occasional gels were
stained with coomassie blue as a check but the majority were
stained with amido black.
Figure 9. Influence of amount and origin of proteins on the Rf of the "skewed"
band in 10% acrylamide gels. Aliquots of proteins from sections
0 to 2 mm, 2 to 4 mm, and 4 to 6 mm behind the root tips of control
pea seedlings 48 hours of age and of a composite sample of equal
amounts of protein from sections 0 to 2 mm and 4 to 6 mm behind
the root tips were subjected to analytical gel electrophoresis.
0.80
0.70
Rf
0.6 0
0.50
100
ZOO
300
400
Lowry protein ( agigel)
500
600
77
Analysis of Electrophoretic Patterns
Presenting data from studies of the electrophoretic behavior of
complex mixtures of proteins from crude plant extracts is difficult.
Spectrophotometric scanning was of little value due to its inherently
limited resolution. Photographic methods suffer from a related dis-
advantage in that critical alignment of the camera lens with a particular portion of the gel is required to observe minor components. In
short, the mechanical methods of gel analysis fall far short of the
resolving power of the eye.
Consequently, I have relied almost entirely on diagrammatic
representations of gels (measured Rf values and estimated band
intensity). In order to show the objectivity of this procedure, I have
included a comparison of diagrammatic, spectrophotometric and pho-
tographic analysis of four gels (Figure 10). The correspondence of
my drawings with the major features in photographs of gels is clearly
seen along with the limitations of the spectrophotometric method.
Steward et al. (1965) and Morris (1966) used 7.5% acrylamide
gels. To facilitate comparison with my results in 10% acrylamide
gels I determined the correspondence of bands in 7. 5% and 10% gels
based on shape, color, intensity and relation to neighboring bands
(Table 2).
Figure 10a, b, c, d. Diagrammatic, photographic, and
spectrophotometric methods of illus-
trating the distribution of soluble cytoplasmic proteins in 10% acrylamide
gels after analytical gel electrophoresis.
78
04
0.5
c.-%1
In
0
0.4
0.3
0.2
0
02
0.4
0.6
0.8
Rf
Figure 10a. Proteins from 1 to 2 mm behind the root tips of
48 hour old control pea seedlings.
1.0
79
II
1
0.6
0.5
o
o
0.4
tr)
C1
0
0.3
0.2
0.1
1
0
I
I
0.2
till III
0.4
of
0.b
0.8
t
1.0
Figure 10b. Proteins from 4 to 5 mm behind the root tips of
96 hour old pea seedlings treated with 5 x 10-5 M
2,4-D between 48 and 50 hours of age.
8
0.6
0.5
o OA
0.3
0.2
0.1
I
0
0.2
0.b
0.4
I
0.8
I
1.0
(Rf
Figure 10c. Proteins from 5 to 7 mm behind the root tips of 120
hour old squash seedlings treated with 5 x 10-5 M
2, 4 -D between 72 and 78 hours of age.
81
NEM
IIIINI
I I
04
0.5
O
0
0
O
0.4
it
0.3
0.2
0.1
I
0
0.2
0.4
0.6
1
0.8
Figure 10d. Proteins from 5 to 7 mm behind the root tips of 108
hour old corn seedlings treated with 1 x 10-3 M
2, 4 -D between 60 and 62 hours of age.
82
Table 2. Correspondence of bands in 7. 5% and 10% acrylamide gels.
Rf in 10% gel
Rf in 7. 5% gel
Corresponds to
0.30
0.48
0.60
0.66
0.16, 0.18 or 0.20
0.73,0.75
0. 54, 0.58 or 0.63
0.91
0.81
0.96
0.96
0.31
O. 38
0.41 or 0.44
Electrophoretic Behavior of Soluble Cytoplasmic Proteins
Proteins from Control Pea Seedlings
The electrophoretic behavior of the protein complement of 48-
hour-old control pea seedling root sections was determined. Pro-
teins were extracted from section 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to
5,
5 to 7, 7 to 9 and 9 to 11 mm behind the root tips and subjected
to electrophoresis in 10% acrylamide gels. As shown in Figure 11,
bands of protein with Rf values of 0.24, 0.28, 0.31, 0.38, 0.48,
0.51, appeared in all sections with constant relative intensity of
stain. Bands with Rf values of 0.12, 0.15, 0.17, 0.36, 0.59 were
also present in all gels but varied in intensity between extracts from
83
I\
im
0 -1
1-2.
2-3
1
3 -4
1
5-7
7-9
9-11
I I
I
0
0.2
I
1
I
0.4
1
0.6
I
0.8
to
(Rf
Figure 11. Electrophoretic pattern of soluble cytoplasmic proteins from
sections 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 7, 7 to 9,
and 9 to 11 mm behind the root tips of control pea seedlings
48 hours of age.
different root sections. Bands with Rf values 0.19, 0.41, 0.71,
84
0.73, 0.84, and 0.96 also increased or decreased in intensity with
distance from the root tip but were not present in all sections. The
most prominent member of the latter group was band 0.41. 3
Some proteins or sets of proteins are common to all sections
of the root while others vary quantitatively with position in the root.
A few proteins are found in certain portions of the root and may be
associated with root development. In order to determine the influence
of seedling age, I examined the protein complement of sections 0 to
1,
1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 7, 7 to 9 and 9 to 11 mm behind
the root tips of seedlings 50, 60, 72, and 96 hours of age. These
seedlings were exposed to 1 x 10-3 M KH 2 PO4 between 48 and 50
hours of age (control treatment). The electrophoretic patterns
differed little from those in Figure 11 suggesting a similar complement of proteins despite differences in total seedling age.
Proteins
were also extracted from sections 10 to 20 mm below the cotyledons
of control pea seedlings 72, 96 and 120 hours of age. The electro-
phoretic distribution of these proteins is depicted in Figure 12.
Band 0.41 was absent in control sections at 72 hours of age but
appeared in increasing quantities at 96 and 120 hours of age. The
intensity of many bands was reduced with increasing age in these
root sections.
3Bands are identified by Rf or the abbreviation: band 0.16
meaning a band with Rf 0.16.
85
age (hours)
I
II
1
II
72
I
96
I
120
0
0.2
0.4
0.6
0.8
1.0
R./
Figure 12. Electrophoretic pattern of soluble cytoplasmic proteins
from root sections 10 to 20 mm below the cotyledons of
control pea seedlings 72, 96, and 120 hours of age.
86
Proteins from 2, 4-D-Treated Pea Seedlings
Proteins also were extracted from sections 0 to 1, 1 to 2, 2 to
3,
3 to 4, 4 to 5, 5 to 7, 7 to 9, and 9 to 11 mm behind the root tips
of seedlings harvested 12, 24, 36, and 48 hours after treatment with
2, 4-D (5 x 10-5 M between 48 and 50 hours of age). The electrophor-
etic patterns of gels from this experiment are in Figure 13. Diagrams of proteins from sections 0 to 1 mm behind the root tip show
most of the bands of protein observed. The remaining diagrams
show only those bands which change with treatment or position in
the root, or that are necessary for reference.
Bands with Rf values of 0.05, 0.24, 0.27, 0.31, O. 38, 0.44,
0. 48 and several others showed little or no changes from control
patterns in Figure 11, Bands 0.12, 0.19 and 0.36, increased in
intensity with time in all sections following 2, 4-D treatment. Bands
0.19, 0.41 and 0.84 were not present in all sections of control seedlings but were observed in all sections of 2, 4-D-treated seedlings.
An increase followed by a decrease in the resolution of bands around
Rf 0.56 to 0.58 was observed following 2, 4-D treatment.
Band 0.32 was seen irregularly in extracts of control seedlings.
It appeared in all extracts of treated seedlings but the intensity was
variable suggesting an artifact. I have included it in some diagrams
only to indicate its occasional presence.
Figure 13a, b, c, d. Electrophoretic pattern of soluble cytoplasmic
proteins from sections 0 to 1, 1 to 2, 2 to 3,
3 to 4, 4 to 5, 5 to 7, 7 to 9, and 9 to 11 mm
behind the root tips of pea seedlings treated
with 5 x 10-5 M 2, 4-D between 48 and 50 hours
of age.
87
0-1
II
I
I
I
I
II
1
1
23
/NM
....,
.....
II
2
3-4
......
.111.
4-5
:--
I
I
I
I
I
1
0
57
7-9
.
4.1..
...mi. .
MN
IIIIIIIIII
0.2
.......
11=1
.
.
.I.
.....;
sm.
0.4
...
0.6
0.8
1.0
Rf
Figure 13a. Electrophoretic pattern of proteins 12 hours after
2, 4-D treatment.
9 -11
88
01
i
1 -2
ll
II
II
7-9
1..0
9-11
0.2
0.6
0.4
0.8
1.0
Si
Figure 13b. Electrophoretic pattern of proteins 24 hours after
2, 4-D treatment.
89
0-1
11
12
=
II
II
II
I
11
5-7
I
II
..
...
79
411111.
I
eall
il
I I
I
IIIIIIII III
0
0.2
04
0.4
0.8
9-11
1.0
a.,
J
Figure 13c. Electrophoretic pattern of proteins 36 hours after
2, 4-D treatment.
90
0 -1
1 -2
1
II
a
MM.
II
II
MO/
4.110
7 -9
11=0
w.1
9-11
MIN
I
I
1111111111
0.2.
0.4
=NM
0.6
0.8
1.0
Figure 13d. Electrophoretic pattern of proteins 48 hours after 2, 4-D
treatment.
91
The most prominent difference between electrophoretic patterns of proteins from control and 2, 4 -D- treated seedlings concerned
band 0.41. In control seedlings it decreased in intensity with dis-
tance and was not found in sections 4 to 11 mm behind the root tip
(Figure 11). Band 0.41 was present in extracts of all root sections
12 hours after 2, 4 -D treatment (Figure 13a). It increased in inten-
sity with time after treatment until it was a moderately prominent
component of the electrophoretic pattern 48 hours after treatment
(Figure 13d). Band 0.41 was in a congested portion of the gel which
prevented adequate spectrophotometric detection, but it was observed in some photographs (Figure 10b).
Incorporation of Alanine-C 14 into Proteins
I measured the uptake of C 14-labeled alanine into root proteins
to define more closely the initial appearance of band 0.41 following
2, 4 -D treatment and to determine if it was present in low levels in
root tips or sections below the cotyledons of control seedlings. Pea
seedlings were exposed to a spray of uniformly labeled alanine-C 14
between 41 and 48 hours of age and again between 50 and 72 hours of
age. About 89% of the radioactivity applied was absorbed between 41
and 53 hours of age and an additional 2% was absorbed during the
next 19 hours. Some seedlings were treated with 5 x 10-5 M 2, 4 -D
while others received only 1 x 10-3 M KH2 PO4 between 48 and 50
92
hours of age.
Root sections of control and 2, 4 -D- treated seedlings were collected according to the following schedule:
Hours since
start of
Seedling
Treatment
Age
(hours)
2, 4-D
of buffer
Number
of
seedlings
treatment per sample
Portion of root sampled
24
24
25
25
25
25
20
20
3-11 mm behind apex
3-11 mm behind apex
3-11 mm behind apex
10-20 mm below cotyledon
3-11 mm behind apex
10-12 mm below cotyledon
6
20
12
15
15
3-11 mm behind apex
3-11 mm behind apex
3-11 mm behind apex
1 Control
2 Control
3 Control
4 Control
5 Control
6 Control
48
54
0
6
60
60
72
72
12
12
7 2,4-D
8 2,4-D
9 2,4-D
54
60
72
24
Only 0. 09% of the radioactivity applied was incorporated into
soluble cytoplasmic proteins. This figure considers only the pro-
teins in a small portion of the plant however, and incorporation into
the total protein of the whole seedlings was probably greater than
indicated.
Changes in the specific activity (adjusted for differences in
alanine-C
14
uptake) of proteins subjected to gel electrophoresis
showed there was continuing incorporation of alanine into proteins
over the course of the experiment (Table 3).
The proportion of
radioactivity precipitated by 50% TCA from the crude supernatant
was larger than anticipated. Alanine of course readily undergoes
Table 3. Specific activity of soluble cytoplasmic protein containing alanine-C 14
from root sections of control and 2, 4 -D- treated pea seedlings.
Radioactivity in
soluble fraction
Treatment
(DPM/ 25 seedlings)
Lowry protein in
soluble fraction
(p.g/ 25 seedlings)
Radioactivity in
soluble fraction
precipitated by
50% TCA (%)
Radioactivity bound
in gel by amido
black (% of applied)
Specific*
Activity
DPM /µg
3
x 103
x 10
1
143
1.41
7.8
6.7
12.9
2
122
1, 15
8.3
7.4
16.9
3
51
1.02
11.5
10.8
23.4
4
100
2.04
11.2
10.0
12.4
5
35
1.31
13.5
12.1
24.2
6
133
1.64
11.9
11.2
17.0
7
234
2.52
12.5
13.0
11.6
8
128
2.40
20.2
16.5
19.6
9
151
2.90
16.1
14.2
13.0
*Specific activity is expressed as DPM precipitated by 50% TCA per p.g Lowry protein adjusted by proportion for differential
uptake of alanine-C14 among treatments.
94
transamination and the resulting pyruvate is susceptible to oxidation.
Large quantities of C1402 may have been lost by this mechanism.
Proteins were subjected to analytical gel electrophoresis. The
destained gels were scanned with a spectrophotometer, sectioned and
counted. The results are expressed as a histogram of radioactivity
(normalized) over Rf superimposed on a tracing of the spectropho-
tometer scan (Figure 14). The histogram of radioactivity represents single observations between Rf values of 0.0 and 0.28 and between 0.55 and 1.0. Average values and the standard error of the
mean from three separate gels are plotted between Rf values of 0.38
and 0.55 (see materials and methods).
There was only a general correspondence between radioactivity
and optical density at 500 nm (0D500). Discrepancies between peaks
of OD 500 and radioactivity around Rf values of 0.08, 0.15, 0.38,
0.62, and 0.75 show some proteins are synthesized or metabolized
more rapidly than others or differ in amino acid composition.
The general level of radioactivity declined with increasing Rf
around Rf 0.41 in all gels except those containing proteins from
2, 4 -D- treated seedlings (Figure 14). Band 0.41 was not preferen-
tially labeled until 12 and 24 hours after 2, 4-D treatment which
means band 0.41 first appeared between 6 and 12 hours after 2, 4-D
treatment (Figures 14c, f, and i). Band 0.41 declined in radioactivity
between 12 and 24 hours after treatment suggesting a more rapid
Figure 14a through i. The distribution of OD500 and C 14 in 10% acrylamide gels following
analytical gel electrophoresis of soluble cytoplasmic proteins from
root sections of control and 2, 4 -D treated pea seedlings exposed to
alanine-C". Seedlings were grown in vermiculite in the dark at
27 deg. C until exposed to alanine-C" between 41 and 48 hours of
age. Seedlings were then treated with 1 x 10-3 M KI-19PO4 containing 5 x 10-5 M chloramphenicol alone (control) or witFi 5 x 10-5 M
2, 4 -D between 48 and 50 hours of age, seedlings were rinsed and
returned to the alanine-C14 treatment solution. Proteins were extracted and subjected to analytical gel electrophoresis. Distribution
of 0D500 in stained gels was measured with a Gilford linear transporter on a Gilford spectrophotometer. Gels were sliced and the
radioactivity in individual slices measured with a scintillation counter. Distribution of CH is based on single observations between Rf
values of 0.0 and 0.28 and between 0.55 and 1.0. Average values
and the standard error of the mean from three separate gels are
plotted between Rf 0.28 and 0.55.
0.6
05
10
04
8
,44
44"-N
O)
O
450
6
a3
0.1
a
I
az
I
0.4
0.8
1.0
(Rj
Figure 14a. Proteins from sections 3 to 11 mm behind the root tips of control
seedlings
48 hours of age.
0.5
5
04
oo
00
Les
=1
10
0.3
0.2
0.1
.11
I
0
I
0.2.
I
I
0.4
I
if
I
04
2
I
0.8
1.0
Figure 14b. Proteins from sections 3 to 11 mm behind the
root tips of control seedlings
54 hours of age.
0.6
0.5
04
a
O
03
0.1
Figure 14c. Proteins from sections 3 to 11 mm behind the root tips of seedlings 54 hours
of
age, 6 hours after treatment with 5 x 10-5 M 2, 4-D.
0.5
0.4
ao
irl
00
0.3
0.2
0.1
(R1
Figure 14d. Proteins from sections 3 to 11 behind the root tips of control seedlings
60 hours of age.
0.5
ONE
10
0.4
44
8
0.3
)5,0
6
4
0.2
el
4
%.1
.4;
0.1
I
0
1
0.2
I
I
0.4
I
Rj
i
0.6
s
a
I
0.8
1.0
Figure 14e. Proteins from sections 10 to 20 mm below the cotyledons of control seedlings
60 hours of age.
0.6
0.5
10
S
O
0.3
0.2.
6
4
0.1
Figure 14f. Proteins from sections 3 to 11 mm behind the root tips of seedlings 60 hours of
age, 12 hours after treatment with 5 x 10-5 M 2, 4 -D.
0.6
10
0.5
0.4
0.3
0.2
0.1
I
0
02
0.4
0.b
0.8
1.0
Figure 14g. Proteins from sections 3 to 11 mm behind the root tips of control seedlings
72 hours of age.
0.6
0.5
0
O
0
0.4
0.3
0.2
0,1
0.2
0.4
0.6
0.8
1.0
Figure 14h. Proteins from sections 10 to 20 mm below the cotyledons of control seedlings
72 hours of age.
0.5
MI
10
04
MI
oLel
c)
0
0.5
a
0.2
FL-if-trite-ifs
.11
0. 1
I
I
0.2.
0.4
0.6
0.8
1.0
'A1
Figure 14i. Proteins from sections 3 to 11 mm behind the root tips of seedlings 72 hours of age,
24 hours after treatment with 5 x 10-5 M 2, 4-D.
104
metabolism of proteins in this band than in adjacent bands.
Proteins from Pea Seedlings Treated with IAA, NAA or Picloram
Pea seedlings were treated with concentrations of IAA, NAA
or picloram sufficient to produce morphological effects similar to
those caused by 2, 4-D (Figures 4b, c, and d). The soluble proteins
were extracted from various root sections 24 and 48 hours after
treatment, and their electrophoretic behavior was compared with
proteins from 2, 4-D-treated seedlings. The electrophoretic patterns
of proteins from sections 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 7,
7 to 9, and 9 to 11 mm behind root tips collected 48 hours after treat,ment are in Figure 15.
The changes in the electrophoretic patterns following treatment
with IAA, NAA or picloram were remarkably similar to those produced by 2,4-D (Figures 13 and 15). Bands 0.12, 0.15, 0.36, 0.41
and 0.84 appeared about the same in extracts from 2, 4-D-, IAA-,
NAA- or picloram-treated seedlings but differed from control seedlings. Bands 0.24, 0.28, 0.44, 0.48 looked the same in extracts
from all treated and control seedlings. When compared to results
with 2, 4 -D- treated seedlings, band 0.31 decreased in intensity and
band 0.38 increased in intensity in seedlings treated with IAA, NAA
or picloram.
Proteins were analyzed from a section 0 to 10 mm behind the
105
0-1 min.
I
Ticlora-nt
1
NAA
IAA
I
0
0.2
04
0.4
0.8
1.0
1-Z ttiltv
iclorain
0
0.2
I 11
NAA
I II
IAA
0.4
0.6
0.8
1.0
Figure 15a. Proteins from sections 0 to 1 or 1 to 2 mm behind the root tips.
106
2 - 3 In m
Ticlorain
I II
II
I
NAA
I
II It II II II
I 11
0
0.2
IAA
.
0.4
0.6
0.8
I
1.0
3 -4 in-n
I
Ticloram
I
NAA
I
I
I
0
IAA
1
I 111111111
0.2
0.4
0.6
0.8
1.0
R..f
Figure 15b. Proteins from sections 2 to 3 or 3 to 4 mm behind the root tips.
107
4-5 won
Tictorcon
0.2
11
NAA
III
IAA
0.4
0.8
LO
Picloramn
NAA
11;11111111
0
0.2
0.4
Rf
04
0.8
IAA
1.0
Figure 15c. Proteins from sections 4 to 5 or 5 to 7 mm behind the root tips.
108
7 -9 innt
TiclOrant
11
NAA
IIIIIII
n111
I
I
1
0.2
0
i
0.4
0.6
0.0
IAA
1.0
9 -11 ittm
1
0
Ticloram
1
0.2
in
NAA
iii
IAA
0.4
0.6
Rf
0.8
1.0
Figure 15d. Proteins from sections 7 to 9 or 9 to 11 mm behind the root tips.
109
root tips of IAA-, NAA- and picloram-treated seedlings 24 hours
after treatment. The electrophoretic patterns were generally intermediate between control seedlings and 2,4-D-treated seedlings harvested 48 hours after treatment. This same pattern was observed
with 2, 4-D-treated seedlings 24 and 48 hours after treatment (Figures
13b and d).
Band 0.41 was present in all sections of roots from IAA-, NAA-
or picloram-treated seedlings 24 hours after treatment. The intensity of band 0.41 increased with time after treatments, and with the
exception of picloram-treated seedlings, was a prominent component
of sections more than 3 mm behind the root tip. The general behavior
of band 0.41 from 2, 4-D- or IAA-, NAA- or picloram-treated seedlings was similar.
Proteins from Pea Seedlings Treated with Ethylene
According to Burg and Burg (1966), the action of some growth
regulating chemicals is mediated by the production of ethylene.
Figure 16 shows the response of pea seedlings exposed to 2,4-D
(5 x 10-5 M for 2 hours) or ethylene (50 ppm for 72 hours). The
morphogenesis of ethylene- and 2, 4-D-treated seedlings was simi-
lar except ethylene-treated seedlings developed a normal complement
of lateral roots rather than the massed lateral roots typical of 2, 4-D
treatment.
The electrophoretic pattern of proteins from a section 0 to 10
Figure 16. Morphogenesis of pea seedlings between 48 and 168 hours
of age. Seedlings were grown in vermiculite in the
dark at27° C. and were treated with 1 x 10-3 M KI-19PO4
alone (control) or with 5 x 10-5 M 2, 4-D between 48 and
50 hours of age or 50 ppm ethylene between 48 and 120
hours of age. The numbers of hours in the figure are
total seedling age from the beginning of the imbibition
period and indicate seedling age at the beginning of the
treatment period and when photographed.
110
CONTROL
48 hours
2,4 -0
44 hewn
CONTROL
4$ hours
24 -0
ETHYLENE
ETHYLENE
RTIMINI
108 hours
72 hours
INI hz.'s
111
mm behind the root tips of ethylene-treated seedlings resembled in
part patterns associated with both control and 2, 4- D- treated seedlings
(Figure 17). The intensity of band 0.41 increased with time, and
after 48 hours of ethylene treatment it appeared the same as a similar band from control root tips or roots extracted 12 hours after
2, 4 -D treatment. After 72 hours, the intensity of band 0.41 re-
sembled that from seedlings 48 hours after 2, 4 -D treatment.
Proteins from Pea Seedlings Treated with 2, 6 -D
Seedlings treated with 5 x 10-5 M 2, 6 -D showed little effect
except for slight stem and root swelling. About 50% of the seedlings
exposed to 5 x 10-4 M 2, 6 -D showed a 2, 4 -D -like response but it was
less intense than would be expected from exposure to 5 x 10-5
2, 4 -D.
M
The other seedlings continued primary root elongation but
had a markedly swollen area where the root tip was located at the
time of treatment. Seedlings which did not continue primary root
elongation showed massed lateral root proliferation which was particularly intense in the swollen region behind the root tip. Seedlings
continuing primary root elongation showed normal lateral root development except in the swollen region which showed 2, 4 -D -like lateral
root proliferation between 96 and 168 hours of age. The electro-
phoretic patterns of proteins from a section 0 to 10 mm behind the
root tips of seedlings continuing primary root elongation after treatment with 5 x 10-5 M or 5 x 10-4 M 2, 6 -D were the same as pat-
terns from control seedlings (Figures 11 and 18). Extracts from
112
age C hours)
I I
II
nn
II
t
0
9b
I
120
111111111
0.2
0.4
f
0.6
0.8
1
1.0
Figure 17. Electrophoretic pattern of soluble cytoplasmic proteins
from section 0 to 10 mm behind the root tips of 96 and
120-hour-old pea seedlings exposed continuously to 50
ppm ethylene from 48 hours of age
113
A
B
C
1
0
0
0.2
0.4
0.6
0.8
1.0
R.4
Figure 18. Electrophoretic pattern of soluble cytoplasmic proteins from sections 0 to 10 mm behind the root tips
of 96-hour-old pea seedlings exposed to 5 x 10-5 M
2, 6-D between 48 and 50 hours of age. Protein were
extracted from sections 0 to 10 mm behind the root tips
of seedlings which continued primary root elongation
after exposure to either 5 x 10-5 M (A) or 5 x 10-4 M
Proteins were also extracted either from
sections 0 to 10 mm behind the root tips of seedlings
which failed to continue primary root elongation (C) or
(B) 2, 6-D.
from the swollen portion of roots which did continue
primary root elongation (D) after exposure to 5 x 10-4
M 2, 6-D. The portion of the root which showed swelling after 2, 6-D treatment was located just back of
the root tip at the time of treatment.
114
roots of seedlings which did not elongate or from the swollen area of
roots which did elongate after exposure to 5 x 10-4 M 2, 6 -D showed
an electrophoretic pattern similar to those from roots of 2, 4 -Dtreated seedlings (Figures 13 and 18). It may be significant that
band 0.41 was prominent in these latter two cases since 2, 4 -D -like
proliferation of lateral roots occurred in these same areas.
Proteins from Control and 2, 4 -D- treated Squash and Corn Seedlings
Electrophoretic patterns of proteins from roots of pea seedlings
which seemed to be associated with stages of development or 2, 4 -D
treatment prompted me to look for similar relationships in corn and
squash seedlings. Distinct differences were observed among electro-
phoretic patterns.
In control squash seedlings, a band with Rf 0.41 was found only
in sections less than 4 mm behind the root tip (Figure 19a). After
2, 4 -D treatment however, band 0.41 appeared in all sections and
showed a marked increase in intensity with distance from the root
tip (Figure 19b). Among several other changes noted, band 0.28
declined sharply from the levels noted in control seedlings. Bands
0.45 and 0.55 declined with distance from the root tip in control
seedlings but were maintained in intensity following 2, 4 -D treatment.
In control corn seedlings, bands 0.21 and 0.30 decreased and
bands 0.25 and 0.28 increased with distance behind the root tip
115
I
I
0-1
II
I
I
I
1
1I
I
I
12
I
I
I
I
I
I
I
I
0
9-11
I
I
I
79
I
I
I
0.2
I
I
I
0.4
I
0.6
I
I
0.8
I
I
1.0
Rf
Figure 19a. Proteins from root sections of control squash seedlings.
116
I
I
1
0-1
I
1
2 -3
11
X
I
1
2
34
I
441...\
44
v3
co
4
45
I
t
X
t
s4
I
57
I
.,.
,..,
79
I I
9 -11
i
0
Figure 19b.
I
0.2
1
1
0.4
1
If
1
0.6
i
t
0.8
t
I
1.0
Proteins from root sections of 2, 4-D treated squash seedlings.
117
(Figure 20a). In 2, 4 -D- treated seedlings the reverse occurred,
bands 0.21 and 0.30 increased and bands 0.25 and 0.28 decreased
in intensity with distance behind the root tip (Figure 20b). Bands
0.05 and 0.15 were found in all sections of 2, 4 -D- treated seedlings
but in only some control root sections. Band 0.41 was of particular
interest. It declined in intensity with distance behind the root tip
of control seedlings although it never disappeared. In 2, 4 -D- treated
seedlings band 0.41 was present in high concentrations in all sections
and increased in intensity with distance from the root tip.
Isolation and Stability of Induced Proteins
The association of band 0.41 with certain meristematic portions
of control roots and its regular appearance in all portions of roots
of seedlings treated with a variety of growth regulating chemicals
is striking. Characterization of the proteins in band 0.41 is desirable because of the possibility of a causal relationship between its
appearance and the initiation or emergence of lateral roots. Toward
this end, I have developed a procedure of isolating the proteins in
band 0.41 which appeared following 2, 4 -D treatment of pea seedlings.
Gentle hand homogenization of the roots in glass in the cold
immediately followed by centrifugation (23, 500 x g for 30 min. ) was
required to prevent degradation of the proteins in band 0.41. The
crude supernatant lost about 50% of the intensity of band 0.41 when
Figure 20a and b. Electrophoretic pattern of soluble cytoplasmic
proteins from section 0 to 1, 1 to 2, 2 to 3, 3
to 4, 4 to 5, 5 to 7, 7 to 9, and 9 to 11 mm behind the root tips of 108 hour old corn seedlings
exposed to 1 x 10-3 M KH 2PO4 alone (control)
or with 1 x 10-3 M 2, 4 -D between 60 and 62
hours of age.
118
0-1
1-2
iir
2 -3
3 -4
it
4-5
I
5-7
it
7-9
9-11
I
0
I
az
I
I
I
0.4
I
0.6
I
I
0.8
I
I
1.0
of
Figure 20a. Proteins from root sections of control corn seedlings.
119
0
1
1 -2
I
I
2-3
SN
.3 -4
44
44
411
tZ1
4 -5
tt
44
5 -7
is I
7-9
9-11
1111111;111
0
0.2
0.4
0.6
0.8
1.0
'Rf
Figure 20b. Proteins from root sections of 2, 4-D treated corn seedlings.
120
stored in ice for 12 hours or at -70 °C. for 21 days.
Storing roots or fresh homogenates in ice or frozen at -200,
-70° or - 196 °C. resulted in an immediate loss of band 0.41.
Cen-
trifugation of the homogenate markedly repressed this loss. Macera-
tion of tissue in Virtis or Waring homogenizers also resulted in rapid
loss of band 0.41 despite immediate centrifugation. These results
suggest that freezing or vigorous homogenization releases degrative
enzymes which are normally bound to cell particulates.
Ammonium sulfate fractionation was useful for stabilization
and purification of proteins on band 0.41. Table 4 shows the pattern
of protein precipitation by ammonium sulfate. Analytical gel electro-
phoresis showed no distinct banding of proteins precipitated by less
than 50% of ammonium sulfate saturation. Porteins in band 0.41
which were recovered from the 50 to 60%, 60 to 75% and 75 to 90%
ammonium sulfate fractions showed little degradation when stored at
-70oC. for 30 days. Ammonium sulfate fractionation is a good means
of preparing large quantities of protein mixtures which contain components of band 0.41.
In another test proteins were extracted from the lower 20 mm
of control and 2, 4 -D- treated pea seedlings. They were fractionated
into proteins insoluble in 50 to 60%, 60 to 75%, or 75 to 90% of am-
monium sulfate saturation and subjected to analytical gel electro-
phoresis. Similar patterns for both control and treated seedlings
Table 4. Protein precipitation by ammonium sulfate.
Protein precipitated
(NH4 )2504
(% of saturation)
Crude supernatant
(mg Lowry protein)
(% of total recovered)
(cumulative % of
total recovered)
109.0
0-20
0.3
0.4
0.4
20-30
0.3
0.4
0.8
30-40
25.0
31.7
32.5
40-50
27.1
34.5
67.0
50-60
14.4
18.3
85.3
60-75
10.6
13.4
98.7
75-90
1.1
1.4
100.0
Total Recovered 78.8 mg
% Recovered
72
122
were observed although the intensities of some bands varied between
treatments (Figure 21). Band 0.41 was prominent in extracts of
treated seedlings showing that the electrophoretic properties of its
component proteins were not influenced by high salt concentrations.
Further purification was accomplished in two steps with a
preparative scale gel electrophoresis apparatus (Canalco Corp. ).
In this instrument electrophoresis continues until the protein moves
off the anode end of the gel and is swept away by buffer. Figure 22
shows the results of a typical preparative run where the buffer which
swept the anode end of the gel was monitored for absorbance at 280
nm and assayed for Lowry protein. The fractions indicated by the
arrows were concentrated and examined by analytical gel electrophoresis.
The order of band "elution" corresponded to the order of migration in analytical gels (Figure 23). Fractions 24 through 32 contained several proteins including those with RE 0.41. In other tests,
fractions from the last half of the peak of OD 500 near fractions 25 to
35 were combined for further purification.
The final purification step was accomplished with a stacking
and sieving dual gel system in the preparative electrophoresis instrument. A small amount of protein moved off the gel in fractions collected between 51 and 57 minutes after "elution" of the tracking dye.
These fractions were combined, dialyzed against homogenization
Figure 21. Electrophoretic distribution of ammonium sulfate fractionated soluble cytoplasmic proteins from sections 0 to
20 mm behind the soot tips of 9 6-hour-o ld pea seedlings
exposed to 1 x 10 M KI-12PO4 alone (control) or with
5 x 10-5 M 2, 4 -D between 48 and 50 hours of age. Proteins were fractionated into groups insoluble in 0 to 50%
(not shown), 50 to 60%, 60 to 75%, and 75 to 90% of ammonium sulfate saturation at 0°C.
123
treatment
control
50 -60
11 I
I
1I
11111 1
1
240
r,
44
Ck
4'
control
60 - 75 k'N
I
I IIIII
1
11
1 11 1 11
1
240
I
1111
I
I
Control
I
I
1
75-90 "
itli II II
1 1111
I
0.2
0.4
1
11
1
0.6
240
1
1
1
I
0.8
I
I
1.0
Figure 22. Elution of OD 280 and Lowry protein from the anode end
of a sieving-stacker single gel in the preparative scale
gel electrophoresis apparatus. The sample contained
the 50 to 90% of ammonium sulfate saturation-precipitable soluble-cytoplasmic proteins which were extracted
from 0 to 20 mm behind the root tips of 2, 4- D- treated
pea seedlings 96 hours of age. Electrophoresis was
conducted at 240 C. at 7.5 ma until the dye front was
within 5 mm of the end of the gel when the current was
reduced to 2 ma. Fractions contained 30 drops of eluate
and those marked with arrows were concentrated and
subjected to analytical gel electrophoresis (Figure 23).
I
200 ,
V
160
120
110
,..)
4-1
CI_
go
r....
C,
40 -)
4
8
12
16
20
24
Tube Number
28
32
36
125
fraction Ituntivr
2
3
4
5
0
0.2
0.4
0.6
0.8
1.0
(R.f
Figure 23. Electrophoretic pattern of proteins from fractions
marked by arrows in Figure 22. Fractions were
concentrated in dialysis bags by dehydration with
polyethylene glycol 2000 and then dialyzed against
homogenization medium.
126
medium and concentrated electrophoretically into a small dialysis
bag. Examination of this final preparation by analytical gel electro-
phoresis showed one major and two minor components (Figure 24).
The spectrophotometer scan was analyzed with a DuPont curve re-
solver which revealed 88% of the area under the three peaks was
contributed by the major component which had an Rf value of 0.41 in
10% acrylamide gels. Complete separation of band 0.41 from other
components probably could be achieved by using larger quantities of
protein or proteins containing radioactive amino acids,
Figure 24.
Diagrammatic, photographic, and spectrophotometric
record of the distribution of proteins isolated from fractions 25 to 35 (Figure 22 and 23) using the stacking and
sieving dual gel preparative scale gel electrophoresis
apparatus. Electrophoresis was conducted at 24 deg. C.
and 12 ma, and 1.5 ml fractions were collected each
minute. A small peak of Lowry protein moved off the
gel between 51 and 57 minutes after passage of the tracking dye. These fractions were concentrated electrophoretically and subjected to analytical electrophoresis
in 10% acrylamide gels.
127
0.6
0.5
o
0.4
4.1
0
0
0.3
0.2
0.1
0
IIIIIIII I
0.2
04
0.4
Rf
0.8
I
1.0
128
DISCUSSION
Morphology of Plants Treated with
Growth Regulating Chemicals
Treatment of pea seedlings with 2, 4-D, IAA, NAA, or picloram
caused inhibition of epicotyl and primary root elongation followed by
emergence of massed lateral roots. Continued exposure rather than
single doses of IAA and NAA were required to produce persistent
effects, however. Eliasson (1961) and Morris (1966) found similar
effects in pea and aspen seedlings, and pea seedlings, respectively.
The response of pea seedlings to 2, 4-D, IAA, NAA, and piclor-
am is understandable in terms of the patterns of metabolism of these
chemicals. Andreae (1967) reported 2,4-D was largely unmetabolized in pea root segments, which accounts for the success of short
term exposure to 2, 4-D in producing persistent effects. In contrast,
IAA and NAA were extensively metabolized in 6 and 16 hours, re-
spectively, in pea root segments. Thus it is not surprising that
long exposure periods were required to produce persistent effects
with IAA and NAA. Picloram resists metabolism in peas but is
translocated towards the top of the plant and is excreted by the
roots 4 which explains why many picloram-treated seedlings resumed
4
Scott, P. C., Research Associate, Oregon State University,
Dept. of Agricultural Chemistry, Personal Communication, Corvallis, Oregon, June 10, 1969.
129
primary root elongation although the epicotyls remained completely
inhibited.
Corn is not sensitive to 2, 4-D for purposes of selective weed
control but my results show that sensitivity is relative, for 1 x 10-3
M 2, 4-D produced morphological effects in corn somewhat similar
to those found in 2, 4-D-treated pea seedlings. Eames (1949) felt
monocots and dicots were affected similarly by phenoxy herbicides
and attributed differences in their external appearance following
treatment to different growth patterns. Botrill and Hanson (1968)
found proliferation of secondary lateral roots in corn seedlings
treated with 2, 4-D. Their pictures of treated seedlings closely
resemble Figure 6.
The mode of action of a chemical may be defined as the series
of steps leading to the overall effect on the organism. Picloram,
IAA, NAA, and 2, 4-D may have a common mode of action in pea
seedlings. Some similarities in the morphological responses of
peas, corn, and squash seedlings to 2, 4-D suggest a common mode
of 2, 4-D action among species.
Anatomy of 2, 4-D- Treated Pea Seedlings
I observed an increased number of pericycle cell divisions
opposite the xylem poles in pea seedling roots 9 hours after 2, 4-D
treatment.
These divisions ultimately led to emergence of massed
130
lateral roots. Scott (1938) and Bond (1948) found the greatest meristematic activity in the pericycle opposite the xylem poles of IAA-
treated pea stem segments and 2, 4-D-treated pea roots, respectively.
They reported few divisions in the cortex or vascular tissue.
-Wilde (1951) and Fisher, Bayer and Weir (1968), working
with 2, 4-D- and picloram-treated bean seedlings, respectively,
and Key and Lin (1966), working with 2, 4-D-treated soybean hypo-
cotyls, reported cell divisions leading to the proliferation of lateral
roots as early as 12 hours after chemical treatment. Colonial bentgrass (Callahan and Engle, 1965) and corn (Botrill and Hanson, 1968)
treated with 2, 4-D also produce massed lateral roots from the pericycle. Eames (1949) found 2, 4-D-induced anatomical changes were
essentially the same in monocots and dicots. Indolebutyric acid,
NAA, and 2, 4-D increased the number of active cell divisions in the
pericycle of Pyrus caucasi suggesting eventual lateral root proliferation (Katulska, 1966).
These reports include research with a diverse group of species
treated with several different growth regulating chemicals. The
similarity of response is striking. It is significant that in every
case sublethal concentrations induced cell divisions in or near the
pericycle over the length of the primary root causing proliferation
of lateral roots opposite xylem poles. In normal seedlings, lateral
roots are also initiated in the pericycle opposite the xylem poles
131
but these centers of activity are widely separated. In other words,
these growth regulating chemicals induced divisions in cells which
have meristematic potential but most of which normally remain
inactive. Thus the morphological response of massed lateral root
proliferation would seem to differ only quantitatively from the norm.
Electrophoretic Behavior of Plant Proteins
Electrophoresis of soluble cytoplasmic proteins from various
root sections of pea seedlings showed some proteins common to all
sections, other proteins which varied in amount from section to section, and a few proteins which were only found in certain sections.
Steward et al. (1965) and Morris (1966) also investigated the electro-
phoretic behavior of proteins from pea seedlings in acrylamide gels.
The extraction and purification procedures Morris (1966) and
I used were nearly the same and from the correspondence of Rf values
in Table 2, our results appear quite similar if Rf values greater than
0.6 are ignored. The justification for ignoring these values is the
presence of the shifting skewed band which interfered with gel interpretation. Both Morris (1966) and Steward et al. (1965) showed bands
of protein which might have changed Rf with increasing distance from
the root tip, which is a characteristic of my skewed band (Figure 9).
My results and Morris' (1966) differ from those of Steward
et al. (1965) probably because of differences in extraction and
132
purification procedures. In particular, Steward et al . (1965) did
not use sucrose or a sulfhydryl agent in their homogenization medium and centrifugation was too slow to sediment the lighter particu-
late fractions. Finally, they concentrated their samples by evapora-
tion in dialysis bags in a stream of air at room temperature. These
conditions are harsh and almost certainly resulted in degradation or
conformational changes sufficient to influence electrophoretic behav-
ior of proteins.
The appearance of band 0.41 was prominent among several
changes in the electrophoretic pattern of proteins from 2, 4 -D-
treated pea seedlings. Band 0.41 was first detected 12 hours after
treatment and increased in intensity with time after treatment and
distance behind the root tip. It was also associated with root tip
sections of control seedlings. Morris (1966) found a band (Rf 0.66
in 7. 5% gels) in extracts of root tips of pea seedlings which also
appeared in all root sections following 2, 4 -D treatment. I do not
know if the proteins I found in band 0.41 (10% acrylamide gels)are the
same ones reported by Morris (1966)(Rf 0.66 in 7.5% acrylamide
gels) but a comparison of Rf values in Table 2 suggests they may be
similar.
Is the apical 10 mm section of roots from 96-hour-old 2, 4 -D-
treated seedlings equivalent to the apical 10 mm section of roots
from 96-hour-old control seedlings? In some respects they are
133
not.
The epidermal and cortex tissues in this section of control
seedlings are younger than in similar sections of 2, 4-D-treated
seedlings. In fact, root tips of 2, 4-D-treated seedlings are more
nearly the same age as sections 10 to 20 mm below the cotyledons
of control seedlings. Further, lateral roots normally emerge from
these latter sections at 96 and 120 hours of age, respectively.
However, differences in the electrophoretic patterns of proteins
from sections 10 to 20 mm below the cotyledons of control seedlings
and from roots of 2,4-D-treated seedlings increased with
time. Significantly, band 0.41 was absent in these control sections
at 72 hours of age but was present in small quantities and large quan-
tities, respectively, at 96 and 120 hours of age. Many other bands
were drastical ly reduced in intensity at 120 hours of age in extracts
from control sections. These differences suggest little biochemical
equivalence between root sections 10 to 20 mm below the cotyledons
of control seedlings and roots of 2, 4 -D- treated seedlings. On the
other hand, similarities in the electrophoretic patterns of proteins
from the lower portions of control and 2,4-D-treated roots suggests
similar biochemical states.
Incorporation of Radioactive Alanine
into Proteins of Band 0.41
Radioactivity in band 0.41 in extracts from 2, 4 -D- treated seed-
lings neither establishes nor excludes the possibility that the
134
appearance of this band represented de novo protein synthesis. This
point can best be established by immunological assay.
The results
do show, however, that band 0.41 first appeared between 6 and 12
hours after treatment and that preferential incorporation of alanineC
14
.
into band 0.41 in control root tips or sections 10 to 20 mm below
the cotyledons did not occur.
Chloramphenicol in the solution of radioactive alanine prevented
the incorporation of radioactivity by microorganisms (Nooden and
Thimann, 1965). The absence of radioactivity in band 0.41 in ex-
tracts from both kinds of control tissues also suggests microbial
activity was not an important source of error.
Proteins from Pea Seedlings Treated
with IAA, NAA, or Picloram
The electrophoretic patterns of proteins from root sections of
seedlings treated with IAA, NAA, or picloram resembled in many
respects patterns from 2, 4 -D- treated seedlings. Of particular in-
terest was the appearance of band 0.41 in extracts of all portions of
these roots 24 or 48 hours after treatment.
Spelsberg and Sarkissian (1966) and Sarkissian and Spelsberg
(1967b) reported sections of IAA-treated bean hypocotyls contained
three new proteins after exposure to 1 x 10-6 M IAA. Exposure to
1 x 10-3 M IAA also led to the appearance of three new proteins but
135
caused the loss of three other proteins as well. These investigator s
froze their sections, however, and did not include a sulfhydryl protecting agent in their homogenization medium. I found these condi-
tions gave poor resolution and patterns suggesting degradation of
protein. This may account for the fact only nine bands were reported
despite application of 400 µg protein per gel. Patterson and Trewavas
(1967) used methods other than gel electrophoresis to show IAA alter-
ation of patterns of protein synthesis.
I found no previous reports of the induction of specific proteins
by NAA or picloram. Based on their known impact on nucleic acid
and protein metabolism, however, it is not surprising to find such
induction (Maheshwari et al., 1966; Esnault, 1965; Malhotra and
Hanson, 1966).
Proteins from Pea Seedlings Treated
with Ethylene or 2, 6-D
Pea seedlings treatedwith ethylene showed morphological effects
similar to those treated with 2, 4-D except proliferation of the massed
lateral roots did not occur. The electrophoretic pattern of proteins
from roots of ethylene-treated seedlings did not show band 0. 41 until
48 hours after the start of treatment. The electrophoretic pattern
resembled in part patterns associated with both control and 2, 4-D-
treated seedlings.
136
Ethylene has been associated with auxin action by some investigators (Burg and Burg, 1966; Abe les and Rubenstein, 1964) while
others have found differences in their modes of action (Andreae et al.
1968).
The lack of proliferation of massed lateral roots in ethylene-
treated seedlings and differences in the electrophoretic patterns of
proteins from ethylene- and IAA-treated seedlings are clear separations of ethylene and auxin effects.
Some 2, 6- D- treated seedlings showed a 2, 4 -D -like morpholog-
ical response. Band 0.41 was present in tissues showing the 2, 4 -D
response and conspicuously absent in tissues which did not show
this response. The recurring association of band 0.41 with the
emergence of lateral roots is striking.
Using gas chromatography, I assayed the 2, 6 -D for 2, 4 -D and
found less than 5% contamination (minimum detectable level). 5 x
10-4 M 2, 6 -D would contain 2.5 x 10-5 M 2, 4 -D which is probably
sufficient to produce visible 2, 4 -D effects. The swelling of roots and
shoots and the appearance of band 0.41 in extracts of certain roots
following 2, 6 -D treatment may have been due to low level 2, 4 -D
contamination.
Band 0.41
I have placed emphasis on proteins in band 0.41 in 10% acrylamide gels. Does band 0.41 represent a real or an artificial part of
137
the protein complement? An unequivocal answer is not possible, but
the shape, color, and behavior and consistent appearance of band 0.41
in treated tissues and some control tissues suggests it contains proteins which are true in vivo components of the seedlings examined.
Sarkissian and Spelsberg (1967a, b) reported a protein which
disappeared from the electrophoretic pattern of IAA-treated bean
seedlings and suggested IAA binding to the protein might have altered
its electrophoretic mobility. All the growth regulating chemicals
used in my experiments could presumably also bind to proteins.
Morris (1966) however, reported no binding of 2, 4 -D with proteins
in 7. 5% acrylamide gels following electrophoresis of soluble proteins
from 2, 4- D -C14- treated pea seedlings.
Macey (1965) reported the release of bound forms of pectin
methylesterase following treatment of artichoke tubers with 1 x 10-5
M 2,4-D. Northen (1942) reported decreases in the structural viscosity of protoplasm following treatment of bean seedlings with IAA
and NAA. Both of these mechanisms could conceivably result in
the appearance of a "new protein in an electrophoretic pattern.
My data on the incorporation of radioactive alanine into proteins is
consistent with the synthesis of new protein in band 0.41 but does not
entirely exclude the mechanisms operative in the experiments of
Macey (1965) and Northen (1942).
The appearance of band 0.41 following treatment with growth
138
regulating chemicals may be the result of de novo protein synthesis,
the release of bound proteins, or alteration of soluble protein conformation and charge. Whatever the mechanism, the appearance of
the proteins in band 0.41 is in close temporal and spatial relationship
with anatomical and morphological events which are triggered by
exposure to certain growth regulating chemicals.
Band 0.41 was found in the root tips of untreated pea and squash
seedlings, in the diminishing quantities with increasing distance be-
hind the root tip of corn seedlings, and in all root sections of seedlings treated with 2, 4 -D, IAA, NAA, or picloram. It was also found
in certain sections of pea seedlings treated with ethylene or 2, 6 -D and
in sections below the cotyledons of control pea seedlings.
The significance of the presence of band 0.41 in all these cases
is uncertain. There is no assurance band 0.41 contains the same
proteins in all cases. Brewbaker et al. (1968), Warner and Upadhya
(1968), Mg.kinen (1968), Mills and Crowden (1968), Alvarez and King
(1969), and others, working with a diversity of plant species, plant
parts, and extraction procedures have all reported the presence of
more than one protein in many single bands following electrophorsis
in acrylamide or starch gels. However, the consistent presence of
band 0.41 in primary root sections which are actively elongating
and in areas from which lateral roots will soon emerge is striking.
The proteins in band 0.41 must comprise a certain minimum
139
proportion of the total protein content in order for band 0.41 to be
visible in acrylamide gels. The protein content was low in root sections near the cotyledons of control pea seedlings; so relatively small
amounts of protein in band 0.41 could make it appear as a major
component of the electrophoretic pattern. In the root tips of control
pea seedlings, however, the protein content was high which means
large amounts of protein must have been in band 0.41 in order for
it to be visible. The appearance of band 0.41 in all root sections of
2, 4 -D -, IAA-, NAA-
.
and picloram-treated seedlings represented
a large increase in the relative amount of the total protein complement in band 0.41. Based on these suppositions, there may be a
relationship between the amount of protein which bands with an Rf
of 0.41 and the magnitude of lateral root proliferation.
The identity and number of proteins which band with Rf 0.41
is unknown, and the possibilities are numerous. However, the possible relationship between root elongation or lateral root emergence
and the appearance of band 0.41 makes the work of Sutcliffe and
Sexton (1968) of interest. They found an excellent correlation be-
tween the localization of Beta-glycerophosphatase and emergence of
lateral roots in developing pea seedlings. Sutcliffe and Sexton (1968)
found this enzyme increased to a maximum in sections 2 mm behind
the root tip and then declined sharply. The concentration of enzyme
increased steadily with distance in sections 10 to 40 mm behind the
140
root tip of pea seedlings (age 120 hours). The p-glycerophosphatase
was localized in the cortical cells of the primary root which bordered
the cells of the lateral root. High levels of this enzyme were also
found in the surface layers of roots where radial expansion was occurring. Fan and Maclachlan (1967) and Datko and Maclachlan (1968)
reported the appearance of large quantities of cellulase and pectinase
in IAA-treated pea seedlings. Their data indicate increased levels
of RNA synthesis preceded the synthesis of these hydrolytic enzymes.
They feel these enzymes are important in the fragmentation of the
walls of expanding parenchyma cells.
I do not know whether the proteins in band 0,41 in any of the
experiments I have done are the same as the enzymes investigated
by Sutcliffe and Sexton (1968), Fan and Maclachlin (1967). or Datko
and Maclachlin (1968). However, sufficient quantities of the proteins
in band 0.41 may be isolated by the procedure I have developed to
permit their characterization.
141
CONCLUSIONS
The hypothesis of this thesis states, "Applying growth regulat-
ing chemicals to plants results in alteration of the interdependent
course of selective protein biosynthesis and morphogenesis." The
objectives were to: (1) Investigate changes in the protein complement
which accompany morphogenesis induced by growth regulating chem-
cials, and (2) to determine whether changes in the protein complement were causally related to morphogenesis.
The hypothesis is partly true and the objectives have been
partly satisfied. Biochemical differentiation was observed using the
resolving power of gel electrophoresis. Qualitative and quantitative
changes were detected in the complement of soluble cytoplasmic
proteins from various root sections of developing pea, squash, and
corn seedlings. The proteins with an Rf value of 0.41 in 10% acryl-
amide gels were of particular interest. Band 0.41 was found only
in the first few millimeters behind the root tips of control pea and
squash seedlings and in diminishing quantities with distance behind
the root tips of control corn seedlings. In pea seedlings, band 0.41
reappeared in root sections near the cotyledons prior to the emergence of normal lateral roots. It seems likely the proteins in band
0.41 are associated with the appearance of normal lateral roots and
the enlargement or elongation of normal primary roots.
142
It may be significant that band 0.41 was found in all root sections of pea seedlings treated with 2, 4 -D, IAA, NAA, and picloram
and in root sections from corn and squash seedlings treated with
2, 4 -D.
The proliferation of massed lateral roots was a striking
morphological response common to all these treated seedlings.
In terms of the original hypothesis and objectives then, this
thesis has shown biochemical differentiation can be observed following application of growth regulating chemicals. It has also shown that,
the timing and location of the appearance of proteins in band 0.41 were
correlated with the emergence of lateral roots in both control and
treated seedlings; however, there is no assurance the two events
were causally related.
Several questions have been raised by this research. Does
band 0.41 from the extracts of control and treated pea, corn, and
squash seedlings contain the same proteins in all cases ? How many
proteins are in band 0.41? What are the proteins in band 0.41, and
how are they related to the morphological responses of pea, corn,
and squash seedlings to growth regulating chemicals? Does the ap--
pearance of band 0.41 represent de novo protein synthesis? If so,
are changes in patterns of nucleic acid metabolism involved?
The
procedure I have developed for the isolation of proteins in band 0.41
can be used as a starting point for answering some of these questions.
143
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APPENDIX
153
APPENDIX
Each stock solution is numbered. Stock solutions used to prepare a particular working solution are identified by name and/or
number.
1.
Homogenization medium
Sucrose
13.72 g
Trizma base (Sigma)
0.242 g
CaCl2
0.033 g
1, 4-dithiothreitol (Cal Biochem)
0. 0015 g
HC1 1 N to pH 6. 9 @ 0°C. dilute to
100 ml with distilled water
2.
Dialysis medium
Sucrose
512.9
HC1 1 N
15 ml
CaCl2
0.441 g
1, 4-dithiothreitol
O. 046 g
Trizma base to pH 7.5 @ 25°C. dilute to 31 with distilled water.
Stock Solutions for Lowry Protein Determination
3.
Na2CO3
2. 0 g
NaOH
4. 0 g
Dilute to 100 ml with distilled water
154
4.
CuSO4 . 5 H2O
0.5 g
NaK Tartrate
1.0 g
Dilute to 100 ml with distilled water
5.
50 ml of 3 + 1 ml of 4 (prepare fresh)
6.
Phenol reagent (Folin and Ciocalteu)
(Hartman and Leddon Co.)
Stock Solutions for Analytical Scale Electrophoresis
7.
Upper acrylamide stock solution
Acrylamide
10.0 g
Bis
2.5 g
Dilute to 100 ml with distilled water
8.
Lower acrylamide stock solution
Acrylamide
30.0 g
Bis
0.8 g
Dilute with water to 100 ml
9.
Upper gel buffer
HC1 1.0 N
24 ml
TEMED
0.1 ml
Trizma base to pH 6.9 at 24°C
Dilute to 100 ml with distilled water
155
10.
Lower gel buffer
HC1 1. 0 N
48 ml
TEMED
0.1 ml
Trizma base to pH 8.9 at 24°C
Dilute to 100 ml with distilled water
11.
Upper reservoir buffer stock solution
28.8 g
Glycine
Trizma base to pH 8.3 at 24°C.
Dilute to 1,000 ml with distilled water
12.
Lower reservoir buffer stock solution
480 ml
HC1 1. 0 N
Trizma base to pH 8.9 at 24°C
13.
Ammonium per sulfate stock solution
0.14 g
Ammonium per sulfate
Dilute to 100 ml with distilled water.
This solution must be made fresh for each use.
Working Solutions for Analytical Gel Electrophoresis
Composition of 7.5% or 10% Analytical Gel
Stock solution
7.5% gel
volume
volume
Lower acrylamide (8)
2.0 ml
3.3 ml
Distilled water
1.0 ml
1.45 ml
10% gel
156
Lower gel buffer (10)
1.0 ml
1.25 ml
Ammonium per sulfate (13)
4.0 ml
4.0 ml
Composition of the Stacking Gel
Stock solution
Volume
Upper acrylamide (7)
2.0 ml
Distilled water
1.0 ml
Upper gel buffer (9)
1.0 ml
Ammonium per sulfate (13)
4.0 ml
Upper Reservoir buffer
Upper reservoir buffer stock solution (11)
100 ml
0.5% bromophenol blue in 95% Ethanol
1 drop
Dilute to 1,000 ml with distilled water.
Lower Reservoir Buffer
Lower reservoir buffer stock solution (12)
100 ml
Dilute to 800 ml with distilled water
Composition of Scintillation Solution
14.
Triton X-100 (Harleco)
1800 ml
Toluene
2560 ml
Vacuum filter through 28-200 mesh Silica gel (Davidson,
grade 14--dried overnight at 80°C in vacuo)
157
15.
Omnifluor (New England Nuclear)
10 g
Toluene
100 ml
Mix 50 ml of 15 with 950 ml of 14 to prepare scintillation
solution.
Common Stock Solutions for Preparative
Scale Gel Electrophoresis
16.
Acrylamide stock solution
Acrylamide
50 g
Dilute to 100 ml with distilled water.
17.
Bis stock solution
Bis
1.2 g
Dilute to 50 ml with distilled water.
18.
Lower Reservoir Buffer stock solution
Trizma base
HC1
121 g
10 N to pH 8 at 24°C
Dilute to 1, 000 ml with distilled water.
Special Stock Solutions for the Sieving-Stacker
Single Gel System Electrophoresis
19.
Gel buffer stock solution
HC1
1. 0 N
TEMED
48 ml
1.2 ml
158
Trizma base to pH 6.9 at 24°C
Dilute to 100 ml with distilled water
20.
Upper reservoir buffer stock solution
Glycine
28.8 g
Trizma base to pH 8.3 at 24°C
Dilute to 1,000 ml with distilled water.
21.
Ammonium per sulfate stock solution
Ammonium per sulfate
0.14 g
Dilute to 100 ml with distilled water.
Special Stock Solutions for the Stacking and Sieving
Dual Gel System Electrophoresis
22.
Upper gel buffer stock solution
1.0N
HC1
TEMED
48 ml
0.46 ml
Trizma base to pH 6.9 at 24°C
Dilute to 100 ml with distilled water
23.
Dual gel elution buffer stock solution
HC1
1.0 N
Trizma base to pH 8.9 at 24°C
Dilute to 1,000 ml with distilled water
480 ml
159
24.
Upper reservoir buffer stock solution
Glycine
144 g
Trizma base to pH 8.3 at 24°C
Dilute to 1,000 ml with distilled water
25.
Ammonium persulfate stock solution
Ammonium persulfate
0.28 g
Dilute to 100 ml with distilled water.
26.
Composition of 10% sieving-stacker single gel
Gel Buffer (1:10 dilution of gel buffer stock
solution (19)
1.5 ml
Acrylamide stock solution (16)
3.4 ml
Bis stock solution (17)
0.71 ml
Distilled water
3.4 ml
Ammonium per sulfate (21)
8.0 ml
Single gel Elution buffer
Gel buffer stock solution (19)
10 ml
Dilute to 1,130 ml with distilled water
Upper reservoir buffer
Upper reservoir buffer stock solution (20)
100 ml
0. 5% bromo-phenol-blue tracking due in
95% ethanol
Dilute to 1,000 ml with distilled water.
1 drop
160
Lower reservoir buffer
Lower reservoir stock solution (18)
100 ml
Dilute to 1,000 ml with distilled water.
Working Solutions for Stacking and Sieving
Dual Gel System
Composition of 15% sieving gel (lower gel)
Dual gel elution buffer stock solution (23)
1.0 ml
TEMED (4.6 ml /ml)
0.5 ml
Acrylamide stock solution (16)
2.4 ml
Bis stock solution (17)
0.15 ml
Distilled water
1.95 ml
Ammonium per sulfate (25)
2.0 ml
Composition of 3. 5% stacking gel (upper gel)
Upper gel buffer (22)
1.0 ml
Acrylamide stock solution (16)
0.56 ml
Bis stock solution (17)
0.2 ml
Distilled water
4.24 ml
Ammonium per sulfate (25)
2.0 ml
Upper Reservoir Buffer
Upper Reservoir buffer stock solution (24)
100 ml
0. 5% bromophenol blue in 95% Ethanol
1 drop
Dilute to 1,000 ml with distilled water.
161
Lower Reservoir Buffer
Lower reservoir buffer stock solution (18)
100 ml
Dilute to 1, 000 ml with distilled water.
Elution Buffer
Dual gel elution buffer stock solution (23)
Dilute to 800 ml with distilled water.
100 ml