SOME EFFECTS OF

2,4-DICHLOROPHENOXY ACETIC ACID ON

THE CARBOHYDRATE METABOLISM

OF ETIOLATED CORN SEEDLINGS

CLANTON C. BLACK, JR.

A DISSERTATION PRESENTED TO THE GRADUATE COUNQL OF

THE UNIVERSITY OF FLORIDA

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA
August, I960

AGRI.

CULTURAI
LIBRARY

AOCJOT/LEDGEMENT

This writer wishes to express sincere appreciation
to Dr. E. G. Rodgers for his untiring help, constant
consideration, inspiring leadership and thoughtful guidance
throughout the writer's entire graduate study; to Dr. T. E.
Humphreys for the generous use of his laboratory facilities,
patient and thorough instructions on unfamiliar techniques,
stimulating discussions and thoughtful guidance in the
course of this research; to Drs . O. C. Ruelke, D. 0. Spinks
and T. W. Stearns, for service on the committee; and to the
Department of Agronomy for financial support during this
graduate study.

The fullest credit is due my wife, who encouraged
the writer to undertake this study, and who worked so
untiringly in a determined effort to complete this step
in our lives.

^^57

i'

DEDICATION

to
my wife

Betty Louise Black

TAiiLE OF CONTEi^TS

Page

ACKNaVLEDGE/VE'JT ii

DEDICATIOiJ iii

LIST OF TABLES vi

LIST OF FIGURES vi i

STATEMENT OF PROr.LE/.l 1

REVIEIV OF LITERATURE 4

Effects of 2,4-D on Gas Exchange

Effects of 2,4-D on -Uneral letabolism

Effects of 2,4-D on the Metabolism of Carbon
Compounds and Related Enzyme Systems

.MATERIALS 24

Plant materials

Preparation of enzyme extracts

EXPERIMENTAL PROCEDURE 28

Studies on the Pentose Phosphate Pathway

Pentose disappearance

Chromatographic studies on pentose disappear-
ance
Assays for specific enzymes
Gl uc OS e-6- phosphate dehydrogenase
6-Phosphogluconate dehydrogenase

Studies on the Glycolytic Pathway

Phosphoglucoisomerase

6-Phosphofructokinase

Aldolase

Glyceraldehyde-3-phosphate dehydrogenase

IV

Page

Phosphoglyceric kinase

Carboxylase

Phosphoglyceric mutase, enolase and pyruvic

kinase
Reagents used in these studies

RESULTS 43

Studies on the Pentose Phosphate Pathway

Pentose disappearance

Chromatograpiiic studies on pentose disappear-
ance
Glucose -6- phosphate dehydrogenase
6-Phospiiogluconate dehydrogenase

Studies on the Glycolytic Pathway

Phosphoglucoisomerase

6-Phosphofructokinase

Aldolase

Glycer aldehyde -3 -phosphate dehydrogenase

Phosphoglyceric kinase

Carboxylases

Phosphoglyceric mutase

Enolase

Pyruvic kinase

DIXUSSION 70

SUA.IARY a:© COiCLUSIOrJS 79

3I3LI0GP^P:f/ 82

LIST OF TAMIS
Table P^ge

1 PE-JTOSE utilizatio:j a:jd fgr:4ation of

HEPTULOSE A:© ilFaOSE 44

2 DISTRI3UTI0M OF CARBOU ATO'-IS FRQ'4 RinOSE-5-

phosphate follov;i-jg 60-.ii:!UTE i;jcunATio:i

PERIOD ^8

3 ACTIVITY OF GLUCOSE-6-PHOSPHATE DE'lYDROGEN-

ASE ^1

4. ACTIVITY OF 6-PlI0SPi-10GLUC0!JATE DEFTi'DROGEIiASE 54

t>. ACTIVITY OF PilOSPIIOGLUCOISQ-.lERASE 56

6. ACTIVITY OF 6-P}I0SPH0FRUCTQKi:JASE 58

7. ACTIVITY OF ALDOLASE 60

8 ACTIVITY OF GLi'CERALDE:f/DE-3-P10SP:^TE

DE:I:DR0GEI1ASE 63

9. ACTIVITY OF EiJOLASE 68

VI

LIST OF FIGURES
Figure f^age

1. Tilt CATA:30LIS4 OF GLUCOSE 3

2. THE REDUCTIOiJ OF TP'A 3Y GLUCOSE -6-PI-10SP:iATE

DEIWDROGEIvIASE ir.' CELL-FREE EXTRACTS ... 53

3. THE REDUCTION OF TPH I'Y 6-PHOSPlIOGLUCaJATE

dehydroge:sIase ih cell-free FXTPjXCTS ... 53

4. ALDOLASE 61

5. THE REDUCTIO:J OF DPH ;:Y GLYCERALDLiuDE-S-

PHOSPIiATE DEiFi'DROGElJASE IM ACETOUE POV/DER
EXTRACTS 61

6. GLYCERALDEif/DE-S-PliOSPl'iATE DE.ri'DROGEI>]ASE . 64

7. PHOSPHOGLYCFRIC Kl:iASE 64

Vll

STATEMENT OF PROBLEM

Copious amounts of research have been directed at
attempting to elucidate the basic mechanism or mechan-
isms of action of plant growth-regulating materials. De-
spite the intensive and persistent efforts of research
workers to solve this problem, the basic mechanism or mech-
anisms of action of plant growth-regulators are not known
(56).

Since 2,4-dichlorophenoxyacetic acid (2,4-D) is one
of the earliest known synthetic plant growth-regulating
compounds, more research has been directed at attempting
to elucidate its basic mechanism of action than for any
other compound. A major area of research on the basic
mechanism of action of 2,4-D is its effects on plant meta-
bolism. Recently, Humphreys and Dugger (80, 81, 82) noted
that 2,4-D affected the catabolism of glucose in etiolated
corn seedlings (Zeg m^y? L.) by increasing the participa-
tion of the pentose phosphate pathway in glucose catabolism
(Figure l). Their Xb viyp work indicated that glucose
catabolism was accommodated almost entirely via the pentose
phosphate pathway in the roots of 2,4-D treated corn seed-
lings. These results stimulated the research in this
dissertation, which v;as undertaken to evaluate the effect

or effects of 2,4-D on the jjn vitro activity of enzymes
extracted from the roots of 2,4-D treated corn seedlings.

The basic hypothesis of this study was that 2,4-D
affected the activity of an enzyme or enzymes of either
the glycolytic pathway or the pentose phosphate pathv;ay
(Figure l) or both, which results in a shift of the major
pathv/ay of glucose catabolism. To test this hypothesis,
the activities of the enzymes of the control, i.e. buffer
treated etiolated corn seedlings, v;ere compared with the
activities of the enzymes of the 2,4-D treated etiolated
corn seedlings. This comparison was made in each experi-
ment in this study.

The studies on the pentose phosphate pathway (Figure l)
consisted of assaying the activities of glucose-6-phosphate
dehydrogenase and 6-phosphogluconate dehydrogenase, and
studying the disappearance of ribose-5-phosphate (R-5-P)
and the appearance of heptulose and hexose. The glycolytic
pathway was studied by assaying for the activities of each
enzyme of the pathway from glucose-6-phosphate (G-6-P) to
pyruvate (Figure l).

starch.

glucose
A

Y

glucose-6-phosphate-

B

K

6-phospho-
gluconolactone

6-phospho-
cluconate

ribulose-5-
phosphate

N

ribose-5-
yphosphate

^0

P

erytlrerose-
4-phosphate

glyceraldehyde;;^
3-phosphate

sedoheptulose- ^

7-phosphate

2 carbon
pool

xyulose-5-
phosphate

Glycolytic enzymes:

A. hexokinase

B. phosphoglucoisomerase

C. 6-phosphofxuctokinase

D. aldolase

E. triose isonnerase

F. glyceraldehyde-3-
phosphate dehydrogenase

G. phosphoglyceric kinase
H. phosphoglyceric mutase
I, enolase

. J. pyruvic kinase

Pentose phosphate pathway enzymes:

K. glucose-6-phosphate
dehydrogenase

:^ fruct OS e-6- phosphate
C

V
fructose-1, 6-phosphate

D

dihydroxy-

acetone

phosphate

1, 3-d iphosphogly cerate

G
3-phosphoglycerate

Y

H

2-phosphoglycerate

I
phospho-enol- pyruvate

Y

pyruvate
krebs cycle

L. gluconolactonase
M. 6-phosphogluconate

dehydrogenase
N. phosphoriboisomerase
0. phosphoketopentoepimer-

ase
P. transketolase

Figure 1. THE CATA130LIS\< OF GLUCOSE.

REVIEW OF LITERATURE

A prodigious amount of research was stimulated when
the hormone types of plant growth-regulators such as 2,4-D
were introduced in the early nineteen-f orties . This growth-
regulator was extensively studied, primarily because it
selectively controlled dicotyledonous plants and because
it was cheap, safe and easy to use. By virtue of its
selectivity, the commercial use of 2,4-D was quickly devel-
oped. Hundreds of compounds have been synthesized since the
introduction of 2,4-D which exhibit varying degrees of
phytotoxicity. 3ut it soon became evident and is more evi-
dent today that very little basis existed for the synthesis
of new compounds and that practically nothing was known
concerning the basic mechanisms whereby these compounds
influenced plant grov/th. V/hile it is true that many plant
growth-regulating substances have been synthesized and
developed to such a degree that they can be used commer-
cially without a knowledge of their mode of action in
plants, it is perhaps equally true that a clearer under-
standing of the modes of actions of the known growth-
regulating substances could assist in making better use
of the known substances and could orient the synthesis of
new substances.

4

A complete review of the literature covering hundreds
of experiments on the effects of 2,4-D on plants demon-
strated several prominent factors. First, a large percent-
age of the data available was on the practical uses of 2,4-D
in agriculture. Second, many of the studies directed at
determining the effects of 2,4-D on plants were concerned
with morphological or anatomical effects produced by 2,4-D
at varying periods up to a year after application. Studies
of this type seem to be concerned with the result of 2,4-D' s
action, but do not seem to be directly related to its in-
itial mechanism of action. Third, some short-time studies,
i.e. some as short as five minutes, showed that 2,4-D has
some effects on plants within a fairly short period of time.
Several workers have advanced the idea that to learn the
basic mechanism of 2,4-D' s action, studies should begin soon
after its application to the plant. This approach seems
logical. Therefore, this review of literature will not cite
many research papers in which the data were collected long,
i.e. several weeks or months, after application of 2,4-D.
Fourth, although numerous hypotheses have been advanced con-
cerning the mode of action of 2,4-D, none have completely
withstood the intensive scrutiny of repeated and extended
research. Fifth, 2,4-D may have several modes of action,
and a search for the mode of action might be futile. Sixth,
several theories as to the mode of action of 2,4-D were

reasonably supported by research data and should be con-
sidered in a study of the possible mode or modes of action
of 2,4-D. ^

After considering the factors cited above, it was
decided to limit this review of literature to research
papers v;hich deal with the probable initial mode or modes
of action of 2,4-D and several physiological responses to
2,4-D.

Effects of 2f4-D on G^s E)^chanqe

Although the effects of 2,4-D on gas exchange by
numerous plants or tissues thereof have been studied
extensively, these studies have not been effective in
determining the mode of action of 2,4-D. Several reasons
exist for this lack of effectiveness. When it is reported
that 2,4-D increases or decreases or does not affect the
oxygen uptake of a particular plant, this is just a general
observation because many areas or sites in plant's meta-
bolism could be stimulated or inhibited by 2,4-D. Thus,
the general effect a worker might record, such as oxygen
uptake or carbon dioxide (CO2) evolution, could be caused
by many reactions within a plant. Numerous other variables
which could cause changes in gas exchange are the age and
type of plant tissues used, concentration of 2,4-D, length
of time tissue is treated, length of time between treatment
and recording of experimental data, pH of the 2,4-D solu-
tion, portion of plant studied, soil moisture supply, and

and prior nutrition of the plant tissue. Because the
gas exchange by 2,4-D treated tissue is dependent upon
such a large number of variables, it is not surprising
that increases (6,16,19,20,34,51,71,78,80,92,116,123,143)
and decreases (77,110,112,147,149,157,162) or no effects
(83,89,114,159) have been observed by many research
workers. In experiments on the effect of 2,4-D on
photosynthesis, similar types of results have been
reported (36,41,47,100,114). Therefore, although the
information from gas exchange studies has been useful
in delineating areas for further study, it has not pro-
vided the answer to the mechanism of action of 2,4-D.

Effects ?f 2^4-D on wlinergl Aetabolism

Similarly, the same variables which influence the
gas exchange from plants treated with 2,4-D also can
influence other aspects of metabolism.

Wolf et al. (165) observed, 14 days after treating
soybeans (Glycine max L.) growing at three levels of
nitrogen {l) with a solution of 20 ppm of 2,4-D, that the
potassium in treated leaves was lower, but that the
potassium level in the entire plant was not affected.
Similar effects were noted when tomato plants (lycffP^r-
sicon e^culentum -Ull) (126,127) were treated with 2-
methyl-4-chlorophenoxyacetic acid (XPA) and tobacco
(Nicotiana 1;abacum L.) with 2,4-D (164). Cooke (33)
stated that within the first 24 hours after treating beans

8

(PI)a^e9).iis vu),qari§ L.) with 2,4-D, the uptake of potassium
was markedly increased, but that after 24 hours this uptake
was markedly inhibited. He postulated that a stimulation
followed by an inhibition could be due to a similar effect
on respiration which might control the uptake of minerals
from the soil. Bass et al. (9) applied 2,4-D to a primary
leaf of cranberry beans and in six days noted a higher
potassium content in leaves and roots, and a lower content
in stems of treated plants than in control plants. An
interesting relation of potassium nutrition to translocation
of 2,4-D was reported by Rice and Rohrbaugh (128). They
noted that low potassium levels in tomato plants inhibited
2,4-D translocation, while increasing the potassium levels
increased translocation.

Treatment with 2,4-D has been observed to inhibit the
accumulation of potassium nitrate (KiJO^) in excised v;heat
roots (JiiiisuTQ spp) (114), and to promote the accumulation
of the salt in sugar beet leaves (ggta vyt^gari? L.) (145).
Hanson and Bonner (67) stated that 2,4-D has no direct
effect on the process of salt uptake, but that it has
three indirect effects: 2,4-D promotes uptake of rubidium
(Rb), which can be related to increased tissue hydration;
pre-treatment with 2,4-D results in reduced salt uptake
which is attributed to a competition between salt and water
for a common energy supply; and pre-treatment with 2,4-D
results in an increased capacity to gain Rb in the initial
hour of absorption. This is interpreted as signifying an

increased cation exchange capacity of the tissue resulting
from increased growth. Wolf et al. (I6b) grew soybeans at
three nitrogen levels. When he treated them at the same
rate, the plants at the highest level were easiest to kill,
and the ease of kill was correlated with the nitrogen
level. This indicates that plants in a high metabolic
state were more adversely affected by 2,4-D. The data of
Asana et al. (4) indicate that per unit volume, the rate of
uptake of nitrogen is not affected by 2,4-D, but that the
total nitrogen uptake may be reduced due to restricted root
growth. Klingman and Ahlgren and Rhodes (126) reported
some results which generally agree with this. Berg and
McElroy (12) and Frank and Grigsby (46) reported 2,4-D
treatment caused an increased nitrate content of certain
weeds and crops, but in other weeds and crops the treat-
ment had no effect,

Rakitin and Zemskaia (121) reported results of treat-
ing the susceptible bean plant in which the nitrogen up-
take is sharply decreased, while in the more resistant
oat (Av^n^ sativa L, ) the nitrogen uptake is only slightly
decreasedo He postulated that this differential response
is an indication of resistance against 2,4-D by oats, and
that cereals are more capable of detoxication of 2,4-D,

The metabolism of phosphorus in 2,4-D treated plants
has been studied extensively due to the key role of phos-
phorus in plant metabolism (107,108). The results of
these studies are quite variable. This variation cf results

10

probably is caused by the same factors which were listed
as influencing gas exchange measurements. Increased
phosphorus uptake was observed (IGO) aiid increased inorganic
phosphorus content of the entire plant (lOl), stems (125)
and roots (9) has been reported. Decreased inorganic
phosphorus content has been reported in whole plants
(126,118) and in the leaves (43,120,125,164) while others
have reported that 2,4-D treatment had no effect on the
phosphorus content of the entire plant (165), leaves (9),
stems (9,43) or roots (43,125). Rohrbaugh and Rice (130)
and Fang and i>utts (43) presented data which indicate that
2,4-D is not readily translocated in phosphorus deficient
plants and that when phosphorus is supplied, the distribu-
tion of 2,4-D and phosphorus follov^s the same pattern.

Despite the contradiction in the results given above,
several papers present results which indicate that a
mechanism of action of 2,4-D is related to phosphorus

metabolism. Fang and Butts (43) demonstrated that the

32

incorporation of p into glucose-1-phosphate and hexo-

sediphosphate was influenced by 2,4-D treatment. Thus,
2,4-D could be affecting phosphorus metabolism or sugar
metabolism, or both. Qrmrod and Williams (113) demonstrated
a striking decrease in the inorganic phosphate content of
soybeans within less than five minutes. Concurrently, the
soluble organic phosphorus increased in the same striking
manner. This work, in particular, illustrates the need for
short time-intervals between treatment and analysis of the

11

plant to deteroiine the initial mechanism of action of
2,4-D.

Cooke (33) noted that the uptake of calcium was en-
hanced initially by 2,4-D treatment, but that within 48
hours the uptake was decreased. Calcium has been reported
to be higher in leaves (165) and roots (164) of 2,4-D
treated plants, although Bass et al. (9) stated that six
days after treatment the calcium content of all tissues
of cranberry beans is lower than that of the controls.

The effect of calcium on cell wall elasticity and
plasticity is of particular interest due to the demonstra-
tion by several research groups that auxin may affect the
activity of pectin methylesterase (PIE) (1,21,60,61,62).
The general theory of the effect of calcium on cell walls
is that the divalent cation reduces wall plasticity by
cross-linking two carboxyl groups (1,48). '>'1ethylation of
pectin is thought to increase wall plasticity by reducing
the number of carboxyl groups which may be cross-linked by
divalent cations (l). Qrdin et al. (117) presented data
which indicate that indole acetic acid (lAA) increased the
formation of methylesters of pectin. These data support
the hypothesis that esterif ication of carboxyl groups of
pectin is involved in the mechanism of cell expansion.
Bryan and Newcomb (21) noted that lAA stimulated the
activity of PME above the control level. Glaszious and
Inglis (60,61,62) presented data which indicate that auxins
are effective in binding P*4E tc cell wall preparations.

12

P'4E theoretically controls the methyl content of pectin
by demethylation; thus, if auxin reduced its activity, an
increase in the total methyl content of pectin could occur.
Therefore, in the presence of auxin, the methyl content of
pectin is increased, and consequently cell expansion is
increased. If this reaction of auxin is a binding of P-IE,
it should be insensitive to metabolic conditions such as
temperature and presence of oxygen. The work of Adamson and
Adamson (l) supports this idea and further substantiates
the theory that auxin-induced cell wall expansion could be
caused by an auxin-induced absorption of PME.

In 1955, Bennet -Clark (ll) noted that the chelating
substance ethylenediaminetetra^cet ic acid (EDTA) would
act as a growth substance by stimulating the extension
growth of Avena coleoptiles. v.'orking independently,
Johnson and Colmer (83,84) and Heath and Clark (68,69)
reported that plant growth substances could act as chelating
agents. They proposed that the growth promoting action of
plant growth substances is through the binding of ions
such as copper, magnesium ( -^g) and calcium. Further
research has generally given support to this theory
(3,23,32,70,85,86), although Fawcett (44) repeated the
work on changes in optical density (O.D.) due to chelation,
and concluded that changes in 0. D. were not due entirely
to chelation. The general status of this theory today is
that chelation could affect certain enzymatic reactions

13

and shift metabolic patterns, thus affecting growth; but
this has not been substantiated and, therefore, does not
eliminate the possibility that the growth-regulating
molecule could have other properties which affect growth.

The effects of 2,4-D on other nutritional elements
have been studied, but no conclusive results have been
reported (9,33,160,165).

Effg£tS-fif...2»4rP.,on.,.the , 'letabsIi^3„.Qf , Caj:b.Qn..CQapgynds
gr)dRelat|ed_EnzYm^ System^

The possibility that auxins affect the metabolism of
carbon compounds and play a role in enzyme activity was
realized in the early research on the mechanism of action
of auxin. In work with Aye pa coleoptile sections, 3erger
and Avery (13,14) noted that the activities of glutamic
isocitric, alcohol and malic dehydrogenases were in-
hibited, enhanced, or not affected, depending upon the
concentration of lAA present. Thus, they postulated a
role of enzyme activator for auxins. Many research
vi/orkers presently are continuing work on this basic idea,
although they do not agree as to what area of metabolism
is affected. Tv;o major areas of research on the mode of
auxin action have developed, one in favor of some phase
of intermediary metabolism and the other in favor of changes
in cell structure and the ensuing water absorption (22).
It seems logical to hold to the idea that some change in
the plant's metabolism could occur initially, and this could

14

influence changes in cell structure and subsequent water
absorption.

The metabolism of nitrogen containing substances in
plants as affected by 2,4-D treatment has been studied
extensively. Although the results are not specific, several
general effects of 2,4-D treatment have been observed which
are reproducible. The total protein content of treated
tissue generally is increased (34,40,49,54,124,132,133,163,
172), but decreases have been noted in leaves (34,49,50),
while simultaneous protein increase in stems and roots of
the same plants resulted in a total increase in protein.
Galston and Kaur (55) fed labeled 2,4-D to etiolated pea
stem (Plgum sativum L.) sections for 18 hours. Centri-
fuged fractionation revealed that the labeled fraction
was localized in the centrifugal supernatant fraction
devoid of all particles. '.■.'hen this fraction was heated,
the protein from treated cells did not coagulate after
ten minutes of boiling, while the untreated proteins
produced a copius white precipitate under the same con-
dition. This effect on proteins correlated closely with
the effect on growth at various concentrations of 2,4-D.
The treatment did not affect the total protein content.
Auxin analogs which did not promote growth were less
effective or completely ineffective in preventing coagula-
tion. The effect was greatly reduced or not produced at
all when growth substances were added in vitro. The
auxin-induced alteration of the physical state of cellular

15

proteins may be ioiportant in explaining auxin action.
Gordon (63) reported some results which indicated lAA
may be associated with proteins as an absorbed, unstably
bound electrolyte.

Studies on the effect of 2,4-D on amino acids have
been inconclusive, with increases (2,54,99,119,124) and
decreases (54,99,119) in free amino acids having been
reported. A partial explanation of this type of results
might be a stimulation of deaminating enzymes as re-
ported by Moewus (113). Akers and Fang (2) exposed
2,4-D treated beans to €^^©2 and found a large increase
in the incorporation of C-"- into aspartic and glutamic
acids. They also noted a decrease in photosynthesis.
Thus, they postulated more CQ2 enters through the Krebs
cycle with a subsequent increase in amino acids. Boroughs
and Bonner (17) found that lAA did not affect the incor-
poration of labeled glycine or leucine into proteins of
corn and Ayena coleoptiles. Luecke et al. (102) reported
that the contents of thamine, riboflavin and nicotinic
acids were decreased in leaves and increased in stems of
2,4-D treated beans. The activity of proteolytic enzymes
in 2,4-D treated tissues has been studied with increases,
decreases, or no effects on their activity being reported
(48,49,50,121,124).

The effect of auxins on nucleic acid metabolism
appears to be a particularly fruitful area of research.
It is generally agreed among physiologists and biochemists

16

that nucleic acids are key components in the control of
grov/th. Skoog (139) presented a good discussion of the role
of nucleic acids in growth and presented a hypothesis link-
ing lAA action with nucleic acid metabolism. He gave
experimental results in which both deoxyribonucleic acid
(D:ja) and ribonucleic acid (RMA) are increased in tobacco
pith tissue treated with lAA (136). Croker (35) studied
the effect of 2,4-D on mitosis in Alliym cepa L. He noted
that 2,4-D affected the nucleic acid cycle in much the same
manner as ionizing radiation. Skoog (139) also noted the
striking similarities in effects produced by chemical
grov/th-regulators and ionizing radiation. As further
evidence of their participation in growth, Rasch et al.
(122) noted that both R'.Vk and D:]A levels increased in
tumor cells from plant tissue. Rebstock et al. (125)
reported that the nucleic acid phosphorus content in stem
tissue from 2,4-D treated plants was approximately double
that of the non-treated stem tissue. West et al. (163)
noted that herbicidal concentrations of 2,4-D increased
R.'-IA content of soybean stem tissue. Other workers have
noted that 2,4-D treatment increased the soluble organic
nitrogen fraction in plant tissues (49,50,166).

Biswas and Sen (15) floated coleoptile sections in
substrates labeled with P^^ ^^ q14 ^^^^^i and without lAA
in a study of the incorporation of labeled compounds. After
tv;o hours the nucleic acid fraction of the sections was
isolated and the radioactivity determined. The activity

17

was taken as an indication of the effect of lAA on in-
corporation and thus the effect of lAA on the cellular

metabolisn. D:ja and RfJA fractions from L\A treated tissue

39

incubated with p^^ counted higher, indicating that lAA

affected the synthesis of nucleotides coTiposing the
nucleic acids. V/hen incubated with labeled acetate,
formate or glycine, lAA treatment did not affect the
counts, lie concluded that lAA stimulates the synthesis
of nucleotides and phosphorylation reactions as evidenced
by the tracer studies; while the compounds labeled with
C , which were all known to contribute to the synthesis
of purine and pyrimidine bases of nucleic acids, were not
stimulated. Thus, the reactions leading to synthesis of
purine and/or pyrimidine ox to sugar moieties of the
nucleic acids were not affected, even though the phos-
phorylation reactions were affected.

Key and Hanson (91), in a study of the soluble
nucleotides of etiolated soybean seedlings, noted that
2,4-D induced a large increase in one fraction eluted from
an ion-exchange column. V/hen this compound was added to
excised root tips or isolated mitochondria, it acted as
an uncoupler of oxidative phosphorylation in a manner
similar to that of dinitrophenol (D\'P), Other workers
have shown that 2,4-D can act as an uncoupling agent of
oxidative phosphorylation (19,147). Aaxie and Forti (104)
states that the primary effect of auxin does not depend
upon phosphate acceptor availability, but more probably

18

involves the activation of oxidative enzymes. French
and Lieevers (51) agree somewhat with this view in their
idea that anabolic reactions catalyzed by 2,4-D stimulate
the use of adenosine triphosphate (ATP) and thus respira-
tion. In further studies, Key et al. (92) found mitochon-
dria from 2,4-D treated tissue to be larger and to have
an increase in phosphorylative and oxidative rates when
compared with mitochondria from untreated tissue. He also
noted that during growth, these mitochondria increased in
acid-soluble nucleotides, phospholipides, and possibly
R'.IA. He concluded that gro'.\rth induced by auxins involves a
grov/th of mitochondria and that this growth is regulated
through nucleotide metabolism.

Very little work has been done on the effect of
2,4-D on lipid metabolism. This lack of research probably
is due to the lack of information on the normal metabolism
of lipids. Key et al. (92) reported that 2,4-D increased
the phospholipides in mitochondria during growth. Weller
et al. (161) reported that the percentage of fatty acids
in bean plants treated with one drop of 0.1 percent 2,4-D
was not affected six days after treatment. The percentage
of total lipids (ether extract) v;as reported by Sell et al.
(131) to be slightly increased in cranberry beans following
treatment with ortho, meta and para-chlorophenoxyacetic
acids. The activity of castor bean (Ricj.nus communis Linn.)
was inhibited as much as 70 percent by 2,4-D (66). Kvamme

19

et al, (97) reported that wheat germ and castor bean lipases
were inhibited by 2,4-D with castor bean lipase being inhib-
ited on the order of 400 times more than wheat germ lipase.

Total starch content in 2,4-D treated tissues usually
is decreased (111,123,132,142,143,151,165,166,167) as are
other polysaccharides (94,132), Only two papers (80,161)
reported that 2,4-D had no effect on the starch or poly-
saccharide content. V/ort and Cowie (168) reported the ac-
tivity of amylase was increased in 2,4-D treated tissues,
although other workers have reported that starch hydrolysis
was inhibited in yitro by 2,4-D (18,151). Neely et al. (U5)
reported that the activity of both alpha and beta amylase
was inhibited by 2,4-D. Salivary amylase was also reported
to be inhibited la vitro by 2,4-D (156), but crystalline
human amylase was reported to be non-responsive to 2,4-D jjj
vitro (45). tost of the work with polysaccharides indicates
that 2,4-D treatment increases their utilization jji yiyp.
It was proposed fairly early in the search for the mode of
action of 2,4-D, that carbohydrates were depleted and the
plant subsequently died (111). Klingman and Ahlgren (94)
stated that at death the carbohydrates would be nearly ex-
hausted, but further research has not substantiated this
theory (123,143).

The changes in sugar content of 2,4-D treated plant
tissues have been reported by numerous workers. Mitchell
and Brown (ill) reported that sugars in 2,4-D treated
morning-glory ( Ippmoea spp) plants increased above the

20

controls at first, but then decreased and were nearly
depleted by the third week folloiving treatments, Using
dandelions (Taraxacum officinale '■'.'. ). Ras-nussen (123)
found that 2,4-D treatment caused an initial rapid in-
crease in reducing sugar content of roots, but that later
the reducing sugar content fell toward the level of the
controls. He concluded that the action of 2,4-D on
dandelion was principally one of destruction of carbo-
hydrate reserves. Smith (142) noted that the amount of
soluble sugars rose slightly by the third day following
2.4-D treatment, then fell steadily. Buckwheat plants
(Faqopyrum esculentum M. ) sprayed with 2,4-D were analyzed
for total sugars by Wort (167). He found total sugar
increased in the stem the first two days but fell below
the controls afterward; total sugar in roots and leaves
declined steadily. Similar results also were reported
later (166).

Both reducing and non-reducing sugars were lower in
stems of 2,4-D treated beans (132) and wild garlic (Allium
vineale L. ) (94). Weller et al. (161) reported that
non-reducing sugars v>/ere depleted in bean leaves and roots
following 2,4-D treatment. Wolf et al. (165) reported
reducing sugars were consistently higher in treated plants.
Two groups of workers reported that reducing sugar content
was not affected by 2,4-D treatment (80,161). Skoog and
Rcbinson (140) incLbated tobacco stem segments with various concontrations

21

of lAA for several months. They noted that reducing sugar
content increased in all cases.

In 1956, Humphreys and Dugger (78) began a series of
experi-nents on the effects of 2,4-0 on plant metabolis-n.
They noted that, although 2,4-D treatment increased the
rate of respiration of etiolated pea seedlings, the respira-
tory quotient of both treated and untreated seedlings
remained near 1.0. These results suggested that carbo-
hydrate v;as the major substrate being oxidized in both
treated and untreated seedlings. In 1957, they (80) re-
ported that the reducing sugar, sucrose and starch con-
tents of 2,4-D treated and untreated seedlings were
essentially the same, thus concluding that the higher rate
of respiration in 2,4-D treated seedlings was not due to
a greater amount of respiratory substrate being present
in these seedlings. The pathways of glucose catabolism in
both 2,4-D treated and untreated root tips of pea, corn
and oat seedlings were evaluated in short-time experiments
by collecting the C O2 evolved when glucose-i-C and
glucose-6-C were supplied as substrates (79). This
work was based on the idea that if glucose were broken
down via the glycolytic pathway, the rate of C ©2
production from the first and the sixth carbon of the
glucose molecule should be the same. If, on the other

hand, glucose were broken down via the pentose phosphate

14
pathway, the rate of C 0 production from the first

carbon of the glucose molecule would initially be greater

22

than that from the sixth carbon. In these experiments,
they found that 2,4-D caused an increase in the amount of
glucose catabolized via the pentose phosphate pathway.
They postulated that 2,4-D increases respiration by
causing more glucose to be catabolized via the pentose
phosphate pathway (80).

They further demonstrated by feeding labeled sub-
strates, i.e. glucose, pyruvate, succinate and acetate,
that both 2,4-D and D;JP promoted catabolisn of exogenous
substrates by blocking synthetic metabolic pathways in
intact etiolated corn root tips (81). Further evidence
of glucose catabolism via the pentose phosphate pathway
in 2,4-D treated tissue was obtained using labeled glu-
cose and labeled gluconate (82). They concluded that in
etiolated corn root tips the catabolism of glucose, when
10 molar (-1) 2,4-D was used, was almost totally
accommodated via the pentose phosphate pathway. Fang
et al. (42) fed labeled glucose to bean stem tissues and
concluded that 2,4-D treatment caused an increase in the
amount of glucose catabolized via the glycolytic se-
quences. The variation in the results of Fang et al.
and those of Humphreys and Dugger could be caused by
several factors: Humphreys and Dugger' s results were
obtained i-nmediately following 2,4-D treatment, whereas
Fang et al. used tissue which had been treated seven
days prior to the study; difference in plant tissues

23

used; and the point made by Humphreys and Dugger (82),
that evaluation of catabolic pathway of glucose based only
on the yield of C O from labeled glucose is not possible.
Thus, the v;ork by Humphreys and Dugger strongly indicated
that 2,4-D treatment increased glucose catabolism via the
pentose phosphate pathway as opposed to the normal glyco-
lytic scheme. These results stimulated the research of
this thesis.

.■^TERIALS

Pj-ant fnateriaJ^s. Corn seed (var. Dixie 18) were
soaked in distilled water 24 hours with aeration and
placed in porcelain trays on moist paper towels. The
trays were covered with a sheet of aluminum foil and
placed in the dark at 22° C for 60 hours. The etiolated
seedlings were divided into two groups and then were
placed in glass microscope slide trays with the roots
down and the cotyledons resting on the microscope
slides. One group of the seedlings was treated by
immersing the roots in phosphate buffer, pH 5.3, lO"^ '4.
The other group of seedlings was treated by immersing
the roots in buffer plus 2,4-D, 10""^ M. The trays were
placed in the dark for 12 hours at 22° C. After a 12-
hour treatment period the seedlings were removed, washed,
blotted dry and the roots excised. The roots from each
group were weighed and used to prepare enzyme extracts.

Preparation of enzyme extracts. Two kinds of ex-
tracts were prepared from each group of roots. Cell-free
extracts and acetone powder exti-cts were prepared from
each group. The same procedure was followed for both
2,4-D and buffer treated roots.

24

25

Ten grams of excised roots were added to 100
milliters (ml) of water (4-6*^ C) and placed under
refrigeration for about 30 minutes. The water was
decanted off and the roots ivere placed in an ice-cold
mortar containing 10.0 ml of 0.05 4 tris (hydroxymethyl)
aminomethane (Tris), pH 7.4 plus 1.0 ml of 1.0 ..1 EDTA.
The roots were ground until no intact roots were visible.
The resulting homogenate was filtered through four layers
of cheesecloth, and the resulting filtrate centrifuged
at 900 X gravity (G) for ten minutes at 0° C.

The yellowish-brown supernatant fraction was de-
canted and its pM adjusted to 7.0 with dilute sodium
hydroxide (.M'aOH) . The supernatant (12-18 ml) then was
placed in cellophane tubing and dialyzed overnight under
refrigeration against two 500 ml portions of 0.01 A
Tris, pH 7.4. The dialyzing solution was changed about
10:00 p.m. each evening. After dialysis, the pH of the
dialyzed supernatant was adjusted to 7.4 with dilute
NaOH. This dialyzed supernatant was designated as a
cell-free extract. Cell-free extracts were used the
same day they were prepared.

The procedure followed in preparation of the
acetone po'.vder extracts is essentially that given by
i^ageman and Arnon (65). Ten grams of corn roots were
added to 100 ml of water (4-6*^ C) and placed in the
refrigerator for about one hour. The water was decanted
off and the roots were ground until no intact roots

26

were visible in an ice-cold mortar which contained 15
ml of 0.1 -A phosphate buffer, pH 8.2, which was 0.03 .A
with respect to F:DTA. Cold acetone (125 ml, -15° C)
was added slowly with stirring to the homogenate. The
resulting slurry was immediately filtered with suction
through Whatman number one filter paper on a Buchner
funnel. The precipitate was washed three times with
75 ml portions of cold acetone (-15° C) and left under
suction until free of an acetone odor. The filter
paper containing the precipitate was placed in a vacuum
dessicator and evacuated with a water aspirator for 15
minutes. The vacuum was released and phosphorus pentoxide
(P20t) was added to the dessicator, a vacuum was drawn
and the precipitate was dried in vacuo for 12 hours.
Then the acetone powders were stored in a dessicator,
over calcium chloride, in the refrigerator.

Acetone powder extracts were prepared by an extrac-
tion procedure which consisted of stirring the filter
paper plus the acetone powder (a fibrous yellow material)
for 15 minutes in 25 ml of a solution which contained
0.01 .A phosphate buffer and 0.0015 M EDTA, pH 7.2. The
resulting slurry was centrifuged at 10,000 X G for five
minutes at -5° C, then filtered through V/hatman number
one filter paper. The resulting filtrate v/as designated
as the acetone powder extract. All extracts were used
the same day they were prepared.

27

The nitrogen content of each extract was determined
by digesting 0.1 ml of the extract in 1.0 ml of sulfuric
acid, followed by Nesselerization. Therefore, the activ-
ity of each enzyme in each extract is presented on a
nitrogen or protein basis. The nitrogen concentration
was multiplied by 6.25 to obtain protein concentration.

EXPERI..tE[vITAL PROCEDURE

Studies on the Pentose Pl)o$ph>a1;e P^thwa^y

Pentose di^app^ar^rjce. Ribose-5-phosphate was used
as the substrate in these studies on pentose disappearance.
Reaction mixtures were prepared, using cell-free and acetone
powder extracts, which contained R-5-P. At the times zero
and 60 minutes, the concentrations of pentose, heptulose
and hexose were determined. To support these studies,
chromatographs were made of solutions prepared from the
reaction mixtures at zero and 60 minutes.

Each reaction mixture used for studying the disappear-
ance of pentose and the appearance of heptulose and hexose
was prepared in a long, narrow test tube in a water bath
at 38 C. The reaction was started by adding the cell-free
or acetone powder extract. Each reaction mixture consisted
of: 0.2ml of triphosphopyridine nucleotide (TP;j) (4 milli-
grams (mg)/ml); 0.1 ml of f lavinadenine mon-nucleotide
(FMN) (1 mg/ml); 0.1 ml of -IgCl^ (O.l ^) ; 3.4 ml of Tris
buffer (pli 7.4, 0.1 l) ; 0.2 ml of R-5-P (O.l A); and 2.0
ml of cell-free or acetone powder extract to give a total
of 6.0 ml.

At the timed increments of zero and 60 minutes after
starting the reaction, aliquots (l.O or 2.0 ml) of the

28

29

reaction mixture were taken and immediately mixed with
equal volumes of 10.0 percent trichloroacetic acid (TCA)
to stop the reaction. The resulting mixture was centri-
fuged at 900 X G for five minutes to remove protein. The
supernatant fraction was used to determine pentose, hep-
tulose and hexose concentrations in the reaction mixture
at the indicated times.

Pentose was determined colorimetrically by the
orcinol method of Bail (8) as modified by viejbaum (109).
When reacted with orcinol, pentoses yield a product with
a maximum absorption at 670 millimicrons (mu) , Heptulose
also was determined with orcinol following the general
procedure of Horecker and Smyroniotis (74). The heptu-
lose maximum absorption occurs at 580 mu. Horecker and
Smyroniotis (74) outlined the procedure followed in the
pentose and heptulose determinations. The concentrations
of pentose and heptulose in an unknown solution can be
calculated from density measurements at 670 mu and 580
mu, which are obtained from the unknown solution and from
known standards of the pentose, arabinose, and the hep-
tulose, sedoheptulosan.

Hexoses were determined colorimetrically by the
method of Dische et al. (37). Ashwell (5) outlined the
procedure followed in these experiments. To determine
hexose, 1.0 ml of the supernatant fraction described
above was added to 4.95 ml of a mixture of six parts of
sulfuric acid to one part of water, cooled, then boiled

30

three minutes, cooled, 0.11 ml of cysteine hydrochloride
added, mixed and allowed to stand for two hours at room
temperature. The hexose concentration then was deter-
mined by obtaining the O.D. readings at 415 and 380 mu
and comparing these O.D. readings with those obtained
from known concentrations of the hexose, glucose.

Samples of known sugars were run in preliminary
experiments to test the reliability of the procedures
described above. In the range of sugar concentrations
used in these experiments, the procedures proved to be
quite reproducible, llorecker and Smyroniotis (74) in-
dicated that fructose interfered with the pentose deter-
mination, but in these preliminary experiments it did
not .

Chromatographic gi^udies yn pentose disappearance.
These chromatographic studies were designed to support the
studies on pentose disappearance and the appearance of
heptulose and hexose. The same reaction mixture used in
studying the disappearance of pentose was used. At the
timed increments of zero and 60 minutes, 3.0 ml samples
of the reaction mixture were removed and placed in a
boiling water-bath for ten minutes. The samples were
cooled and then incubated at 38 C for 30 minutes with 1.0
ml of alkaline phosphatase (l mg/ml), pH 9.5, to hydrolyze
the phosphate sugars. The samples again were placed in a
boiling water-bath for ten minutes. The samples were

31

centriguged and the supernatant fraction was decanted off
and evaporated with heat and vacuum to about 0.3 ml vol-
ume. The solution was mixed with two ml of absolute
ethanol and evaporated to dryness. The resulting pre-
cipitate was dissolved in 0.5 ml of water and used as the
sample for studying sugars chromatographically .

The filter paper (Schleicher and Schuell, number
589 blue) used in these ascending chromatographic studies
was pre-washed with oxalic acid. From 10 to 50 lambda
spots were made, one inch from the bottom of the paper.
Large sheets of filter paper (18 X 24 inches) were used.
These sheets were formed into cylinders and were equili-
brated for at least six hours by suspension over the sol-
vent, prior to placing them in the solvent. The solvent
system used was n-butanol-pyridine-water in the ratio
3:2:1.5 for one-dimensional chromatographs . V/hen two-
dimensional chromatographs were run, water-saturated
phenol was the second solvent. The orcinol spray of
Klevstrand and :Jordal (93) was used for ketoses, particu-
larly sedoheptulose, while the B-naphtholamine and phos-
phoglucinol sprays given by Smith (144), were used to
detect other sugars. Identification of the sugars in
the ethanol solution was made by comparing their Rf values
and colors with those obtained from known sugars. The
known sugars were processed using the same procedures
given above for the unknov/n sugars.

32

Assays for specific enzymes. The procedures described
in the remainder of this dissertation will be concerned
with assays for the activity of specific enzymes. In
assaying for each enzyme, preliminary experiments, not
reported in this dissertation, were run, in which the
limiting concentration of enzyme was determined for each
reaction. It was considered that the range of limiting
enzyme was obtained when the amount of enzyme could be
doubled to result in the amount of product being doubled
in a fixed time interval.

G,;.ViCflsgr^rBh9§Pl]ia^e..dghydy9g?nase. The presence of
this enzyme was studied by measuring the reduction of
TPN at 340 mu with a Beckman DU spectrophotometer, with
G-6-P as the substrate in the following reaction:

G-6-P + TPI4^^6-PHOSPHOGLUCO:mTE + TPNH-I- tit

The assay mixture contained the following components:
2.0 ml of Tris buffer (pH 7.4, 0.1 J) ; 0.1 ml of TPN
(4 mg/ml); 0.1 ml of -IgCl (O.l .l) ; 0.1 ml of G-6-P
(O.l -0; cell-free extract; and water to 3.0 ml total
volume in i eckman corex cells of one centimeter (cm)
light path. TPM was omitted from the blanks, and the
reactions were started by adding the extract. The in-
crease in O.D. was followed at timed intervals for
periods up to 30 minutes.

33

6-Phosphoqluconate dehydroQenase . The presence of
this enzyme was studied by measuring the reduction of TP;J
at 340 mu, employing the same procedure as was used in
assaying for G-6-P dehydrogenase. The same assay mix-
ture was used except 0.1 ml of 6-phosphogluconate {6-PGA)
(0.1 A) was used as the substrate in the reaction given
below:

6-PGA ^ TPif-^^RIBULOSE-5-PHOSPmTE + CO^+TPiiH +H .

S^y^ies on the GlvcQlYtic P^lM^

Phosphoqlucoisomerase. A colorimetric method, based
on the color formed by fructose-6-phosphate (F-6-P) in
the presence of resorcinol (129), was used to determine
the F-6-P formed in the reaction below:

G-6-P^ — ^F-6-P.

F-6-P gives about 65 percent of the color of free fructose
in this procedure (141). A typical reaction mixture
consisted of: 0.2 ml of Tris buffer (pH 9.0, 0.05 0;
0.2 ml of G-6-P (0.1 ■') ; and 0.1 ml of cell-free extract.
The mixtures were incubated for ten minutes at 38 C. The
reaction was stopped by adding 3.5 ml of 8.3 .A ICI. One
ml of 0.1 percent resorcinol in 95 percent ethanol was
added and the mixture heated for ten minutes at 80 C,
then cooled in a water bath at room temperature. The color
intensity then was read with a Klett colorimeter using
filter number 54 (500-570 mu). After determining the

34

limiting enzyme concentration, the reaction mixture given
above was incubated for periods of 10, 15 and 20 minutes,
and the micromoles (umoles) of F-6-P were determined by
calculations from the Klett readings of known samples of
F-6-F.

6-Phosphofructokinase. 6-phosphofructokinase, which
catalyzed reaction 1 below, vjas measured by the procedure
of Ling et al. (98), using the complete system sho'.vn in
equations 1, 2 and 3 below. In this system, F-6-P and ATP
are substrates, and -Ig is an essential co-factor.

1. F-6-P -I- ATP^-^F-1,6-P + ADP.

2. F-1 G-P^^DI.'^r/DRC&CYACrTONE PH0SP:IATE4-

GL -CLRALDEir/DE-3-PI iOSPf lATE .

3. Dlif/DROXYACETOiJE PHOSPIiATE -f- DPNH < >-ALPhlA-
GL'i'CHROP:iOSPHATE + DP:Jt

The fructose-1, 6-phosphate (F-l,6-P) formed in reaction
1 is cleaved by aldolase, and the dihydroxyacetone phosphate
which is formed is reduced through the utilization of reduced
diphosphopyridine nucleotide (DP.JIi) to alpha-glycerophosphate.
The oxidation of DP?JH in reaction 3 was followed with a Heck-
man DU spectrophotometer at 340 mu, as a measurement of the
activity of 6-phosphofructokinase. A typical reaction mix-
ture in a Beckman corex of one cm light path consisted of:
0.5 ml of tris buffer (p!i 8.0, 0.2 l) ; 0.3 ml of ATP (0.1 ".) ;
0.2 ml of F-6-P (0.1 l) : 0.3 ml of lgCl2 (O.Ol .1); 0.1 ml of
cysteine-hydrochloride (0.2 4); 0.1 ml of DP:JH (2 mg/ml); 0.1ml

35

of aldolase (1 mg/Til); 0.05 ml of alpha-glycerophosphate
dehydrogenase (1 mg/Tjl); 0,1 ml of cell-free extract; and
water to 3.0 ml total volume. The blank consisted of the
complete reaction mixture as given above, minus DPNIi. The
reactions were started by adding F-6-P, The endogenous
activity of the reaction mixture without the extracts also
was determined. This activity was subtracted from the
measurements obtained with the extract to give the activity
of 6-phosphofructokinase in the extracts.

Aldolase. Aldolase activity was measured by the meth-
od of Sibley and Lehninger (134) as modified by 3eck (10),
in which the triose phosphates formed in the reaction below
are trapped with hydrazine:

F-l,6-P^-^DIHYDRCXYACETaJE PHOSPHATE +
GLYCERALDEir/DE-3-PHOSPIiATE.

The triose phosphate hydrazones formed were treated
with alkali, and tlie color produced by the addition of
2,4-dinitrophenylhydrazine (2,4-DiIPH) and NaCSi was read
with a Klett colorimeter using filter number 54, A
typical reaction mixture consisted of: 1.0 ml of Tris
buffer (pll 8.6, 0.1 l); 0.25 ml of F-1, 6-P (0.05 ..1);
0.25 ml of hydrazine (0.22.0; 0.2 ml cell-free extract;
and 0.8 ml of water. The reaction mixtures were incu-
bated at 38*^ for ten minutes with various amounts of
extract. Results of the assay are presented in a graph

36

of ml of extract versus Klett readings per mg N to
demonstrate a typical experimental result. The absolute
amounts of triose phosphates were not determined, but
since their concentration is proportional to the Klett
readings, the Klett readings are taken as an indication
of enzyme activity.

Glyceraldehyde-S-phosphate dehydrogenase . Activity
of glyceraldehyde-3-phosphate dehydrogenase (GPD:!) could
not be demonstrated in cell-free extracts; therefore,
acetone powders prepared following the basic method of
Mageman and Arnon (65) were used to study the activity
of this enzyme. GPDil was measured in a Deckman DU
spectrophotometer by the optical test of Warburg and
Christian (158) based on the increased 0,D. of reduced
DP?^ and TPM at 340 mu in the reaction below:

GLYCERALDEFri'DE-3-PHOSPllATE -4- DPlf + H^PO.^ ^

1,3-DIPHOSPlIOGLYCERATE -I- DPNH + H^ . "^

The following reaction mixtures were prepared in eckman
corex cells of one cm light path: 0.2 ml of DPM (4 mg/Til);
0.1 ml of sodium arsenate (0.17 l); 0.1 ml of potassium
flouride (O.l M) ; 0.15 ml of reduced glutathione (O.l M) ;
0.1 ml of glyceraldehyde-3-phosphate (G-3-P) (40 mg/6 ml);
1.5 ml of Tris buffer (pH 8.5, 0.1 M) ; 0.02 ml of acetone
powder extract; and water to 3.0 ml total volume. Sub-
strate was omitted in the blanks. Glutathione and G-3-P
were prepared fresh daily. Reactions were started by

37

adding either the extract or substrate. The increase
in O.D. at 340 mu was followed at timed intervals. Results
are presented on the basis of the change in O.D. per mg
U versus time.

Pho?phoqlyceric kins^s^. The activity of phospho-
glyceric kinase was determined by the procedure of
Axelrod and Banduski (7) in which the 1,3-diphospho-
glycerate (1,3-DPGA) formed from 3-phosphoglyceric acid
(3-PGA) in the reaction below, was trapped by hydroxyl-
amine, forming hydroxamic acid:

3-PGA + ATP ^— ^1,3-DPGA + ADP.

The hydroxamic acid formed a colored ferric complex when
reacted vjith ferric chloride, which was read with a
Klett colorimeter using filter number 54. A typical
reaction mixture consisted of: 0.4 ml of a solution of
hydroxylamine (2.5 M) plus MgCl (0.015 0; 0.5 ml of
3-PGA (0.052 4); 0.2 ml of ATP (O.l -0; 0.15 ml of Tris
buffer (pH 7.4, 0.05 m) ; and 0.05 ml of cell-free extract.
The blank consisted of the same reaction mixture, except
3.0 ml of a ferric-chloride (FeCl„) -FCL-TCA mixture was
added prior to adding the extract. The reaction mixtures
were incubated at 37 C in a water-bath for periods of
10, 15, 20 and 25 minutes. The reactions were stopped
by adding 3.0 ml of the FeCl^-HCl-TCA mixture.

38

Carboxylase. When the enzymes phosphoglyceric
mutase, enolase and pyruvic kinase were studied, pyruvate
was measured as the product. If carboxylases are present,
pyruvate could undergo one or all of the reactions given
below (137,15b):

1. CH^* CO • COQH< >"C1L' Ch'O -I- CO^

2. CIL- CO • COOl! -h CIL- CHO^-^CIL- CO • CHOH • CIU +
CO^ J J ^

3. 2 C!I • C.HO < ^CH^- CO • CHOH • Cll^.

Obviously, if pyruvate reacted in this manner when pyruvate
was measured, a stable product was not being measured, but
rather a substrate for another reaction. Thus, if carboxy-
lases are present in the extracts, pyruvate can not be
measured to indicate the activity of phosphoglyceric mutase,
enolase and pyruvic kinase. Therefore, carboxylase activity
was determined manometrically by CO2 evolution in the
presence of pyruvate, as the substrate following the method
of Singer and Pensky (137,138). The following components
were placed in standard 15 ml conical Warburg vessels, and
CO- was checked by the direct method as given by Umbreit
et al. (152): in the main compartment, 1.0 ml of succinate
buffer (pH 6.0, 0.2 M) ; 0.6 ml of 1, l-dimethyl-3,5-diketo-
cyclohexane (0.05 -l) ; 0.1 ml of thiamine pyrophosphate
(ThPP) (5.8 X 10""^ .1); 0.2 ml of :.1gCl2 ^°-°^ "^^ ' ^'^ ^^
serum albumin (one percent); cell-free or acetone powder
extract; water to 2.8 ml total volume; and in the side arm.

39

0.2 ml of sodium pyruvate (0.5 .4). At zero time, the
pyruvate was tipped into the main compartment and CO^
evolution was followed for periods up to two hours.

Pho^phoqlvceric mutgise^ enolase and pyruvic kinase.
This group of enzymes catalyze reactions 1, 2 and 3,
respectively, below:

1 . 3-PH0SPH0GLYCERATE-^-^2-PH0SPH0GLYCERATE .

2. 2-PHOSPHOGLYCERATE^-^PliOSPHO-ENOL-PYRUVATE-l- H^O.

3. PHOSPHO-ErJOL-PYRUVATE 4- ADP-^^ PYRUVATE + ATP.

In assaying for these enzymes it was reasoned that each
one could be studied, if the necessary co-factors and
substrates were added, by assaying for pyruvate. Pyruvate
was determined in these studies by the method of Friedemann
and Haugen (52) as modified by Kachmar and Doyer (87).

Phosphoglyceric mutase activity was assayed using the
following reaction mixture adapted from Grisolia et al.
(64): 0.2 ml of 3-PGA (0.375 A); 1.5 ml of Tris buffer
(pH 7.4, 0.1 .4); 0.2 ml of MgCl^ (O.l -l) ; 0.2 ml of ADP
(0.1 .1); 0.1 ml of 2,3-diphosphoglycerate (2,3-DPGA)
(30 mg/5 ml); 0.1 ml of KCl (0.5 .4); 0.2 ml of extract;
and water to 2.5 ml total volume. Phosphoglyceric mutase
activity was measured in both cell-free and acetone powder
extracts .

Enolase activity was assayed in the following reaction
mixture by utilizing the activity of a phosphatase in the
extracts, which would hydrolyze phospho-enol-pyruvate (PEP)

40

to pyruvate: 1.5 ml of Tris buffer (pH 7.4, 0.1 .l) ;
0.2 ml of 2-phosphoglycerate (2-PGA) (O.l -0; 0.2 ml
of . -19012 (0.1 M) ; 0.3 ml of cell-free extract and water
to 2,5 ml total volume. 2-PGA was made fresh daily.
Using 0.2, 0.3 and 0.4 ml of extract to start the reactions,
the reaction mixtures were incubated ten minutes at 38 C.
Pyruvate was determined after the reactions were stopped
with 2,4-DiMPH. The results of colorimetric determinations
of pyruvate are presented on the basis of Klett readings
per mg .M versus ml of extract added.

Pyruvic kinase activity was measured by the procedure
of Kachmar and Boyer (87), in which the activity of the
enzyme is based on the rate of formation of pyruvate. A
complete reaction mixture had the following components:
1.5 ml of Tris buffer (pH 7.4, 0.1 -l) ; 0.2 ml of PEP
(0.05 .4); 0.2 ml of -VlgCl^ (O.l 4); 0.2 ml of ADP (0.1 A);
0.1 ml of KCl (0.5 a); 0.1 ml of extract; and water to
2.5 ml total volume. The reaction mixtures were incubated
for ten minutes at 38° C. Reactions were started by adding
enzyme extract and stopped by adding 2,4-Dl'JPi!. The blank
consisted of a complete reaction mixture minus PEP, vjhich
was added after the 2,4-D:jPH. The reaction mixtures then
were assayed for pyruvate. 4cCollum et al. (105,106)
noted that reaction mixtures minus ADP were active, indicat-
ing the possible hydrolysis of PEP by a phosphatase, as
illustrated in the following reaction:

PEP -f H^O^-^ PYRUVATE + IMORGAfJIC PHOSPHATE.

41

Phosphatase activity v;as determined in both acetone
powder extracts and cell-free extracts by omitting ADP
from the reaction mixture given above. The endogenous
phosphatase activity was subtracted to obtain the pyruvic
kinase activity.

Reagents ugi^d in these studies. The chemicals used
in these studies were obtained from the following sources:
2,4-D from the Eastman Kodak Company; ribose from Eastman
Organic Chemicals; F-l,6-P from '-lann Research Laboratories,
Incorporated; F-6-P and sodium pyruvate from Nutritional
Biochemicals Corporation; DP'A and DP'I14 from Pabst Labora-
tories; R-5-P, G-6-P, ATP, G-3-P, 3-PGA, 2,3-DPGA and
ThPP from Schwarz 3ioresearch Incorporated; and TPM, TP'JH,
6-PGA, F-6-P, ADP, PEP, 2-PGA and Fl'J from Sigma Chemical
Company^ Sedoheptulosan was generously donated by Dr.
Nc K, Richtmyer of the National Institute of Health, to
whom the author is indebted. The enzymes used in these
studies were obtained from the following sources: aldolase
from '^nn Research Laboratories, Incorporated; alkaline
phosphatase from Nutritional Biochemicals Corporation; and
alphaglycerophosphate dehydrogenase from Sigma Chemical
Company. Bovine serum albumin was obtained fram 'lann
Research Laboratories, Incorporated.

The 2,4-D was neutralized with IJaOH and the sodium
salt recrystallized twice from a water-alcohol solution.
The following compounds were obtained as barium salts and
converted to potassium salts by the addition of a slight

42

excess of potassium sulfate to a solution of the barium
salts in dilute acid: G-6-P, R-5-P, F-6-P, F-l,6-P,
2-PGA, 6-PGA, 2,3-DPGA and 3-PGA. Monobarium DL-glycer-
aldehyde-3-phosphate diethylacetal was converted to
DL-G-3-P by mixing with and aqueous suspension of Dowex
50, heating the suspension and centrifugation to remove
the resin. PEP was obtained as the silver barium salt
and converted to the sodium salt by tituration with a
slight excess of ICl followed by the addition of a bare
excess of sodium sulfate (87) . The other chemicals used
were the highest grades commercially available and were
used without further purification.

RESULTS

Studies on the Pentose Phosphate .Pathway

Pentose disappearance. The enzymes v/hich catalyze
the utilization of pentose and the formation of heptulose
and hexose were active in extracts from both treated and
untreated tissue. In Table 1, all experiments indicated
that pentose was utilized and heptulose and hexose were
formed in the 60-minute incubation period.

Experiments 1, 2, 3, 4 and 5 demonstrate the
disappearance of pentose and the appearance of heptulose
and hexose in cell-free extracts which were made from the
roots of control and treated corn seedlings. Experiments
6 and 7 were included to demonstrate the same system in
acetone powder extracts, in untreated cell-free extracts
and in untreated cell-free extracts with 2,4-D added to
the reaction mixture. Obviously cell-free extracts
(Experiments 1, 2, 3, 4, 5 and 7) were much more active
than acetone powder extracts (Experiment 6) in utilizing
pentose and in forming heptulose and hexose.

In Table 1 it should be noted that the results of
each experiment are presented on the basis of the number
of umoles per six ml and on the basis of the number of
umoles per mg ;J per six ml. The percent change due to
2,4-D is based on the number of umoles per mg "J per six ml,

43

44

CQ

*
UJ
CO

O
X

1X1

Q

<

CO

o

O

O

M

H

U-,

Q
<

o

M

H

M
-J
M

CO
O

w
a.

o a

0)
tn
o

0) o

O -H

a, 4j

T-t,<

96ueq3 o/

/f; BiLi/saxotun

tuj 9
/saxoujn

a-t7*2 oq. anp

a-t7*3 o:^ anp

Tuj 9
/[' 5iu/saxoujn

TUJ 9
/saxoLun

^^q.uau]q.Eaa:x

jaquirif: :;uaujTjadx3

a-t? 3 o^ anp i

I

a6ueq3 c^ 1

XUJ 9 {

/f. 6uj/saxoujn i

XUJ 9 j
/saxouin

o
o

(A

D

a

CO

O

3

'sT -J

rH n

• o

I

I o

t rH

I

I

I

I

I

I a

10

00 -H

I o

I •

I lO

I r-i

I

I

I 07

I D
I

I o

I •

I CM
I CM

I r-i
I

I 10
I D

I o
I

I Csl
I lO
I
I

I V)
I 3

I Q. I Q.

CO ';!■

CM CO

n \0

ON CO
CO 't

c^co

-HCO

I O
I
I o

I Csl

I

I

I

I

I

I o
I •

I CM

I CO

I

I

I

I

I

3

I o
I

I CO
I
I
I

I o
I 3

I .H

CO -H CO CO
Or-* CM lO

O CO*

CO

d

O --i

CM "^

Oro

OCM

I O

I

I

I

I

I

I

I

I

CO

I Oi I Q. I a. I a.

o o

^ a\

coo

lOCO

vO CO

CM CO

coco

-H'cr

CM CO

CMOJ

c^ >H

• •

iD 00

• •

t •

^CO

• •

• •

O

1 o

1 o

I o

•

1

1 •

1 •

o

1 r-

1 o

• o

.— 1

1

1 r-
1

1 lO

1

(/)

1 to

1 to

1 to

D

1 3

1 D

1 3

r-i

1 rH

1 rH

1 rH

I a

CM CO

CM ^

I o
I

I o

I CO

I

to
3

I CL I O. I Q. I CL

CviO
o'cM

• •

O CO

M

M

M

M

M 1

41 Q

<D Q

0) Q

oo

<l;Q 1

^-* 1

<4H 1

1+-I 1

l^H 1

<+^ 1 1

tw ^

tWr;t

14-. TJ-

t!^

'^vf 1

3 "^

3 .»

3 ».

3 •«

3 .^

CQ CM

ra CM

a CM

CO CM

■■:0 OJ 1

rH

CM

ro

•^

tO 1

45

1 o

1° '1

1

1 •

01

0)

1 CO

1 CO 1

M H

-P

1 CO

1 1

Cl> 0) -(J

ro

•

1

1 i

i+^JT C

x:

""•>

1

1 1

MhH <!*

a

1 Ui

1 (A I

3 O

05

lO

1 3

1 3

X! C

0

1

1 •-•

1 >-\ 1

• O

x:

0

t o.

1 a '

Tris
6 ml
he c

a

X

OOJ

'^ iT)

4J

Csl

ro

• •

• •

• •■4-1

t

•

O-H

n CO

"^^ o cr>
3 C

0

CO

4-1

r^ o

OJ n

o e c

M

0

• *

• •

r-t 3 -H

Oj

oo

CN<N

1 O
1 •

1 c^

.^0 M

(N> a>

cnraxJ

x:
+j

•H

x:

4->

c
0

•H
-t->

ro
M

1 • 1

1 .H
1

1 OJ
1 3
1 rH

1 a

100 ug;
give a to
prior to

05
(H

3
0

x:

c

0
c
0
0

ro

a;

OvO

CX)0

; 50^

c

•H

3

• •

• •

1 "^ -PCO

4-1

C

oo

lO o

i [X. CO

xs

•H

! •*->

0)

ro

+J

! ••• 0 +J

+->

C

O lO

r- 'cr

! cr> rD rt)

ro

Q

O

• •

• «

1 3 M

(U

^

u

oo

ro 't

1 0 4-" 05

0 X 01

1 00 <D 4->

s

.H

1 3

05

0

1 o

1 O

^4-1 C

cr>

•

-H

1-U

1 •

1 •

1 r: O'H

CQ

,-1

1 CO

1 lO

1 a, e

■H

1

05

C3

I vO

1

1 H-H

-H^

M

<■

1

1

1 CO

XJ

•^

3

H

1

1 05

vD

QJCM

+->

1 U)

I 3

1 ..0

a>

X

1 3

1 C

1 XI • M

05

■=?

•H

1 -i

1 -rH

0) CvJ 0

s

1 a

1 e

! C 4-4

1 -1-0

cro

Mt

c

1 ro C X»

•

00

0

1 +J n3 (U

0

0 —1

•H

^CN

"=t r-

1 C +J

>

•

+J

• •

• •

1 0 ••• fT>

0

-D

05

05

0

CO lO

in ^

1 0-^ X3X>

a>

3

+J

ro

.-H»-<

1 3 3

ro

-fJ^H

0

a>

1 <D 0

ro

Q.

ro

M

) MO C

c

.-«

M

^ »H

O "^

1 3CM-H

<L

0

U

+J

(U

• •

* •

1 -*->

>

•H

Ol

X

x:

<N -sT

o^<:>

1 X »>a)
1 -Ha. M

■ H

05

0)

4-"

1 £ 1 a>

3

M

0

1 iD 5

0)

X3X!

a>

4J

1 c 1

(H

.-1

XJ

*

1 OcC oj

ro

0

U

->

x>

*

1 -H di

05

1

0

0

a>

$

1 •+->•«(-!

3

>-

a

X5

1 O"- 3

0)

ro ro

x>

* *

-a XI

1 05 3 +J

Xi

•

0)

ro

* *

Qi (U

1 0) X

<D

1

lO

c

* *

+J +J

1 M 0 -H x:

0"

0

Q

O (T3

) -^ f5 +J

a)

rn

+->

1

(U 0)

a;cO

M

a

<u

"^

1+-1 1

M M

\ JZ CM-.

x:

0

r

i+-t ^

-t-> 4-»

1 1— -. 0

0

I-

w^

<

CM

3 e.

C C

1 * ';r •-•

*

M

*

*

n CM

DD

1 • +J

05

*

a>

*

*

1 r^ 0

C

4-«

*

*

ro

0

4-t

*

tr 0)

•H

3

vO

r-

Q.M 4-'

^

46

The reaction mixtures contained 20 umoles of pentose per
six ml at time zero. The pentose utilized (Table l) in
the 60-minute incubation period varied from 2.4 umoles
(txperiment 6) to 13.4 umoles (Experiments 3 and 5). The
formation of heptulose per six ml varied from 1.9 umoles
(Experiment 4) to 5.3 umoles (Experiment l) in the cell-
free extracts. Acetone powder extracts (Experiment 6)
formed only a small amount of heptulose. Hexose formed
per six ml with cell-free extracts varied from 1.7 umoles
(Experiment 3) to 4.3 umoles (Experiment 4). Acetone
powder extracts (Experiment 6) formed 0.9 umoles of hexose,

The umoles of endogenous hexose at zero time was
subtracted when necessary. A correction for hexose at
zero time was necessary only in four instances, because
the extracts were dialyzed to remove sugars and other
soluble substances. The greatest amount of hexose found
at zero time was 0.08 umoles. At zero time, heptulose
was not found in the reaction mixtures.

Treatment of corn roots with 2,4-D resulted in an
increased utilization of pentose (Table l), with the
increase ranging from 8 to 32 percent in Experiments
1 through b. The formation of heptulose also v/as in-
creased from 7 to 70 percent by 2,4-D treatment. The
hexose formed was increased from 8 to 122 percent. Thus,
in cell-free extracts from the roots of 2,4-D treated
corn seedlings, the utilization of pentose and the form-
ation of heptulose and hexose was enhanced.

47

TPN was added routinely to the reaction mixture
although, theoretically, it should not be required to
form heptulose and hexose from pentose. In work pre-
liminary to the results reported herein, the effect of
TPiNi on the reaction was studied. ;Jo effect was noted,
with or without TP:J, on the formation of heptulose and
hexose; but since the general procedure of Clayton (31)
was being used, TPN was added routinely. From the work
with G-3-P presented later, no GPDH activity could be
demonstrated in cell-free extracts, which supported the
idea that the TPN in the reaction mixture was not
necessary.

As an indication of the completeness of the pro-
cedures used in assaying for sugars, the number of
umoles of carbon atoms utilized as pentose and used to
form heptulose and hexose was calculated from Table 1.
Table 2 contains this accounting of the umoles of car-
bon atoms utilized as pentose and found in heptulose and
hexose, following the 60-minute incubation period. In
the buffer treated extracts, from 53 to 99 percent of
the carbon atoms are accounted for in hexose and hep-
tulose, with an average of 79 percent. This 79 percent
of the carbon atoms compares well with the average of
84 percent in untreated tissue (Experiment 7). In the ■
2,4-D treated extracts, from 74 to 97 percent of the
carbon atoms were accounted for as hexose and heptulose,
with an average of 84 percent. This 84 percent accounting

48

c

0

X}

M

(T> fH

U 0

^-^

-^

(TJ T3

+j 0)

00

00

00

00

00

00

00

0 -M

• •

• •

• •

• •

• •

• •

• •

•

H C

CM r-

OvO

cmo

CO 00

-H '^r

lOCO

CM lO

1^

tn D

00

C>00

coco

lOiO

r- r-

CO •^

r^o

*

i^-i s 0

iD

000

1

4J 0

0

^<C <

^

X

x>

Ul

a;

CO

H

-p

•

<e

c

CO

a.

3
0
0

^ CO

^.°.

lO lO

00

CO vO

00 vO

C>Tt

0

COM

O*C0

coco

CM 0

lO r~

r-r-(

CO CM

0

r-\

CM

rHrH

i-t

rH

X*

nj

c

PuQ

C
D

0

lOM

+»

UJ 3

1 vO

CO

S

M

to a,

c

M

-p

O

•H

0

c

a ^

^H

0)

MO

W

u

cC •— 1

e

0

TfvO

0 CO

CM-*

vOOO

•^vO

OJ'^

CM 00

c

^H

0

+J 0)

• •

• •

• •

• •

• •

• •

• •

0

O^

+j

w

^00

iDvO

0 CO

CMiO

r- CO

•^ iT)

CO ro

u

<

-o 0

'H'H

r-^r-K

'-\(^

rHCM

-H ^

fHrH

cdD

0) X

.H

UL. y

c

(/) a>

(0

0

DX

c

CO t-i

XI

•H

r?

N

«+4

O ui

n]

e

f-H

0

M

<0

<5

0

4-«

1+-1 0)

0

3 •-*

0

w

+»

0 0

r-M

-HCvl

CO cH

COvO

CO CO

0 lO

Csl CO

§6

5 vO

(0

-fj^

• •

• •

• •

• «

• •

• •

• •

a>

a>

3

00 r-

0 CM

r--0

COvO

00

Oco

CMO

M

-H

TD +J

CM CO

CO CO

CNCO

^CM

CM CO

Csl rO

3

C)

0

a> 0.

+»

o

e

w) 0

X

ti. r^

0

D 3:

•

•H

Om

M

^

6

>•

1 u

go

1 •'^

0)

a>

c

O-l

-^

(n

i—j

0

*~*'p^

XJ 0

ja

•H

S£

0) 4J
N C

if)iO

lOO

00

lOO

00

OtO

lOO

03

•

■p
0

CO

•H 0)

• •

• •

• •

• •

• •

• •

• •

W)

(0

M

rHO,

CO r-

lO r~

\0 f^

00 CM

CO r-

CMO

0 [^

0)

■M

a>

2:

•H

■^ lO

'^ lO

^ vO

•<^vO

lT) vO

rHCM

sr ^

0)

0

03

u

io

D 03

*

M

0

M

*

0)

4J

-p

Q

*

*

* *

*
*

-O-D

-P

X

01

73

c

* *

* *

-P -M

X

•H

0)

T3

(1>

u

u

u

u

u

a»Q

03 03

e

T3

03

s

a>Q

(D Q

OjQ

<UQ

<UQ

Oj 0)

•

2

+J

4-4 1

•^ 1

l4-« 1

l+H 1

4-1 1

H-i 1

U M

c

— )

0

w

fO

i+-iTt

^-^

iw^

l4-.r;r

^^

4h^

+J-i->

0

a

ro

<u

D ..

3 ..

D »>

D .»

3 *

D .V

C C

•H

0)

3

u

"^ CM

CO CM

:rj CM

ra CM

:a csl

"3 CM

DD

+J

-H

<D

H

0

03
0)
M

XI
03
H

C
0
+J
0)

9

-P

(U

0

OJ

c

M

0)

<

%

<l>

0

CO

*

'

*

*
*

*
*

*
*

H Q>

'H

CM

CO

•<t

lO

vO

r-

(U SX

a.s

X

1 1

-2

X

49

of carbon atoms is the same as the average of 84 percent
with untreated tissue (Experiment 7).

Acetone powders were not as active as cell-free
extracts, and the carbon atoms were not as well accounted
for as in the reaction mixtures with cell-free extracts
(Experiment 6, Tables 1 and 2).

The variability of the results reported in Tables
1 and 2 was a matter of concern. Several factors which
may have contributed to this variability are given below.
First, there was the inherent difference in separate
batches of seedlings. Second, an intermediate compound,
R-5-P, of the pentose phosphate pathway, a complex system,
was used as the substrate. R-5-P and its products can
undergo many reactions to form a large number of sub-
stances which were not measured in these studies. At
zero time the pentose measured was R-5-P, but at 60
minutes, the pentose could be any or all of the following:
R-5-P, ribulose-5-phosphate, xyulose-5-phosphate or
xylose-5-phosphate. The variability in heptulose and
hexose concentrations may be explained on the basis that
they are intermediates in a complex system. Third, it
should be noted that when the concentrations of sugar
were placed on a basis of per mg N, the amount of variation
changed. In the buffer treatment in Experiment 4, the poor
accounting of carbon atoms could be explained by the
probable formation of three and four-carbon sugars and the
two-carbon component glycoaldehyde, none of which were measured.

50

Chromatogrgphic studies on pentose disappearance .
These studies were designed to support the studies on the
disappearance of R-5-P. Chromatographic studies following
the 60-nninute incubation period demonstrated the presence
of ribose and sedoheptulose in the reaction mixtures.
Known ribose samples had an Rf value of 0.57 and developed
a pink color when sprayed with n-naphtholarnine. One
unknown sugar was identified as ribose by virtue of a
similar Rf value and pink color. The known sedoheptulose
sample had an Rf of 0.55 and developed a blue color when
sprayed with orcinol. An unknown sugar was identified
as sedoheptulose by virtue of its Rf value of 0.52 and
the development of a blue color when sprayed with orcinol.

GJ,ucose-6-ph9spha1;e dehydrogenase. Glucose-6-
phosphate dehydrogenase was found in extracts from both
treated and untreated tissues. The activity of glucose-
6-phosphate dehydrogenase is given in Table 5 on the basis
of the change in O.D. per mg protein per five minutes,
as measured with a i^eckman DU spectrophotometer at 340 mu.

Spectrophotometric assay for this enzyme was com-
plicated by the formation of 6-PGA v^hich v/as the substrate
for 6-phosphogluconate dehydrogenase which also reduced
TPIJ, and thereby increased the observed O.D. values. The
observed O.D. values were used to calculate the maximum
amount of 6-PGA which could be formed in the reaction time.
In a separate experiment, this amount of 6-PGA was used
as the substrate in the same reaction mixture. Only a

51

TABLE 3
ACTIVITY OF GLUCOSE-6-PHOSPIi^TE DEH^i'DROGEi^SE-x-

Experiment Treatment** Change in O.D. per mg % Increase
■Nlumber Protein per 5 min. Due to 2,4-D

8 Buffer 0.70

2,4-D 0.90 28.6

9 Buffer 0.64

2,4-D 0.86 34.4

10 Buffer 0.92

2,4-D 1.06 15.2

•^Incubation mixture consisted of: TPM, 400 ug; MgCl2,
10 uM; G-6-P, 10 uM; Tris buffer, pll 7.4, 200 uM; 0.2 ml of
cell-free extract; and water to 3.0 ml total volume. O.D. read-
ings at 340 mu.

**See Table 1.

52

slight change in O.D. could be detected; therefore, the
presence of 6-PGA and 6-PGA dehydrogenase was not con-
sidered in calculating the activity of glucose-6-phosphate
dehydrogenase.

The activity of glucose-6-phosphate dehydrogenase
was enhanced by 2,4-D treatment from 15.2 to 34.4 percent
over the activity in buffer treated extracts (Table 3).
Figure 2 includes a graph of typical data showing the
activity of glucose-6-phosphate dehydrogenase in cell-free
extracts from both 2,4-D and buffer treated tissue. Cuffer
treatment resulted in an average change of 0.75 in O.D.
per mg protein per five minutes, v^hile the 2,4-D treat-
ment resulted in an average change of 0.94, thus indicating
that Xn yi;^ro G-6-P is oxidized faster following 2,4-D
treatment .

6-Pho^ph9qluconate dehydrogeogse. This enzyme was
active in cell-free extracts from both buffer and 2,4-D
treated tissue. Table 4 contains the results of assays
for this enzyme on a basis of the change in O.D. per mg
of protein per five minutes, and the results of a repre-
sentative experiment are presented in Figure 3.

In assaying for this enzyme, aliquots were used from
the same cell-free extracts employed in assaying glucose-
6-phosphate dehydrogenase. In all experiments, the activ-
ity of 6-phosphogluconate dehydrogenase was higher than
that of glucose-6-phosphate dehydrogenase. The buffer
treated extracts averaged an .D. change of 0.98 as

53

in

-X

1 1 1 1 1

9 ^ y

1

-

-

o

c
J

1

1 1 1 1 I

\

in

in

o in o ID o

ro CM pj — — .
N Buj/(nuJ0fr£)AilSN3a IVOIidO

Q

<n

N 6iju/(nuJ0l7E)AilSN30 "IVDIldO

5^UJ C

CU(J) O

2 +J

(JL, U4 O

tf>

O O fO
O 0)

r: cc: M

OQ

H = ar

CJ UJ H ft)

Q J3

U4 U4 • n3

o

cr: Hto H

"5

1X1 X O C

J

•ZZCU<-'-^

LU

Hc/) cd

2

OH C

1-

~ X <i>

• a, uu >

<N 1 -H

lO

souu a>

0) 1 UJ

cr»0 1

•HO ►J a»
U^D-J U

_J U-l D

> r^ -H

ca M s

54

TABLE 4
ACTIVITY OF 6-PHOSPHOGLUCOIJATE DEHiDROGEr^lASE*

Experiment Treatment** Change in O.D. per % Increase
Number mg Protein per 5 min. Due to 2,4-D

11 Buffer 0.89

2,4-D 1.15 29.2

12 Buffer 0.88

2,4-D 1.11 26.1

13 Buffer 1.17

2,4-D 1.26 7.7

^Incubation mixture consisted of: TPN, 400 ug; .4gCl2,
10 u4; 6-PGA, 10 u4; Tris buffer, pH 7.4, 200 u^.; 0.2 ml of
cell-free extract; and water to o.O ml total volume. O.D. read-
ings at 340 mu.

**See Table 1.

55

compared to 0.75, and the 2,4-D treated extracts averaged
1.17 as compared to 0.94. The increase in O.D. of 6-
phosphogluconate dehydrogenase over glucose-G-phosphate
dehydrogenase was 0.23 in both 2,4-D and buffer treated
extracts. Thus it is evident that in vitro, 6-PGA is
oxidized faster in 2,4-D treated extracts than in buffer
treated extracts.

These in yitr.Q studies have demonstrated that treat-
ment of corn seedlings with 2,4-D prior to preparing
cell-free extracts results in a general enhancement of
the activity of the pentose phosphate pathway. This
enhancement is evidenced in an increased utilization of
R-5-P, an increased formation of heptulose and hexoses
from R-5-P, and an increased rate of oxidation of both
G-6-P and 6-PGA in cell-free extracts from 2,4-D treated
corn seedlings.

St-udies of the Glycolytic Pai^hway

Phos phogluc ois omer a s e . Phosphoglucoisomerase was
active in cell-free extracts from both buffer and 2,4-D
treated corn roots (Table 5) . The number of umoles of
F-6-P produced from G-6-P by phosphoglucoisomerase per
mg of nitrogen is given in Table 5, Although 2,4-D
lowered the umoles of F-6-P produced in all three experi-
ments, this decrease was not more than seven percent after
20 minutes in the highest case (Lxperiment 14). These
decreases art easily within the range of experimental error.

56

TABLE b
ACTIVITY OF PHOSPHOGLUCOISO-.IERASE-**-

Experiment Treatment** 0:4 flf-Fr^-F PJ:Qdy<;ed pej mg -^
Number 10 min. 15 min. 20 min.

14 Buffer 63.4 97.6 117.1
2,4-D 60.9 82.6 108.7

15 Buffer 55.5 116.7 138.8
2,4-D 66.7 88.9 133.3

16 Buffer 52.8 72.0 107.2
2,4-D 50.0 60.0 101.9

*The reaction mixtures were composed of: Tris buffer,
pH 9.0, 10 u..^, G-6-P, 2.5 uM; 0.05 ml of cell-free extract;
an^ to 0.5 ml total volume with water. These were incubated at
38 C for the prescribed periods of time.

**See Table 1.

57

Therefore, from the results of these in yiiro studies with
cell-free extracts, 2,4-D treatment does not affect the
activity of phosphoglucoisomerase.

6-Ph9sphofructokinase. Quantitative measurements of
the activity of 6-phosphofructokinase were obtained by
incubation of F-6-P, ATP and .'AgCl^ in the presence of
aldolase, DPNH and alphaglycerophosphate dehydrogenase.
6-phosphofructokinase was active in cell-free extracts
from both untreated and treated tissues (Table 6). The
endogenous activity of the reaction mixture was deter-
mined and used to calculate the activity of 6-phospho-
fructokinase. By subtracting the endogenous activity of
0.06 per minute from the activity of the reaction mixture
including the cell-free extracts, the activity of 6-phospho-
fructokinase in the extracts was determined. Table 6
presents the results of these experiments, corrected for
endogenous, on the basis of the change in O.D. per mg
;j per minute.

Treatment with 2,4-D lowered the activity of 6-phospho-
fructokinase as compared to the activity of the enzyme in
cell-free extracts from buffer treated roots. The inhibi-
tion of activity ranged from 7.0 to 12.7 percent. Although
this inhibition appeared to be small, it was consistently

observed.

Aldolase. Aldolase activity in cell-free extracts
was measured by trapping the triose phosphates formed
when F-l,6-P is cleaved by aldolase. In extracts from both

58

TABLE 6
ACTIVITY OF 6-PHOSPiIOFRUCTOKIiIASE*

Experiment Treatment** Change in O.D. per % Decrease
"lumber mg M per min.*** Due to 2,4-D

17 Buffer 0.63

2,4-D 0.55 12.7

18 Buffer 0.86

2,4-D 0.80 7.0

19 Buffer 0.55

2,4-D 0.48 12.7

^Complete reaction mixture contained: Tris buffer, pH 8.0,
100 u'.l; ATP, 30 u.1; F-6-P, 20 al; ..•lgCl2, 3 u-l; cysteine-hydro-
chlorid, 20 u.l; DPiJII, 220 ug; aldolase, 10 ug; alpha-glycero-
phosphate dehydrogenase, 50 ug; and water to 3.0 ml in corex
Beckman cells with 1 cm light path. O.D. readings at 340 mu.

**See Table 1.

■***Corrected for endogenous.

59

control and treated roots, aldolase activity was indicated
by the formation of triose phosphate hydrazones (Table 7).
Figure 4 pictures the results of a typical experiment, and
Table 7 summarizes the results of each experiment. Aldo-
lase activity in cell-free extracts from 2,4-D treated
roots was consistently decreased about 12 percent as com-
pared to extracts from buffer treated roots.

GJ,ycer^ldehyde-3-phosphate dehydrogenase . Attempts
to demonstrate GPDIi activity in cell-free extracts were
not successful; however, acetone powder extracts from the
roots of both buffer and 2,4-D treated seedlings contained
a DPM-dependent enzyme. The lack of a TPiM-dependent dehy-
drogenase is in agreement with early work (65) which failed
to demonstrate this enzyme in etiolated plant tissue, or
tissue which lacked chlorophyll (59), and with the obser-
vation of :4arcus (103), that TPM triose phosphate dehydro-
genase is controlled by the photomorphogenic reaction.

Neither DPl^li nor TPI^IH was oxidized in incubation
mixtures containing all of the constituents used to study
the reduction of DPN.

Neither cysteine nor glutathione v;as necessary as
a co-factor in the reaction. Other workers have reported
that one or the other was necessary (59,96,146,153). The
only effect noted when these were included in the reaction
mixtures was that the O.D. readings more constantly pro-
duced a straight line. Glutathione was added routinely

60

TABLE 7
ACTIVITY OF ALDOLASE*

Experiment Treatment** Klett Readings*** % Decrease
;Jumber Due to 2,4-D

20 Buffer 69

2,4-D 60 13

21 Buffer 66

2,4-D 59 11

22 Buffer 67

2,4-D 59 12

*The following reaction mixture was incubated at 38 C for
10 min., stopped with 0.75 ^j UaOH, and the triose phoshpate
hydrazones :neasured: Tris buffer, pi! 8.6, 100 u.4; r-1, 6-P,
12.5 u''1; hydrazine, 55 u-.l; enzyme extract; and water to total
volume of 2.5 ml.

**See Table 1.

***Change in Klett readings per 0.1 mg of protein added to
the reaction mixture.

61

lOQ

N 6uu|0/(nuuof£)AllSN3a HVOIldO

0) c

c

CO C

X
o

o

I--

I

o

OJH
H

0)

H

U4
CO

8

<

D

cn-4-»

•H 4-'

ox:
M -P
a
o

4-1 +j
o

-o
cr> a>

e-o
-o

CL u) x:
a> o

C 03
M

4->
X

0)

c

•H

c
>

(0

j3

5

M

4->
X

.H I

(1)

H

1+^ e

a>'
u

c
o

ii3n»

62

although it was not required. Table 8 shows the results
of these experiments and Figure 5 presents a graphical
representation of a typical experiment.

The activity of GPDH was decreased from 12 to 20
percent by 2,4-D treatment as compared to extracts from
buffer treated corn root. The rates of these reactions
were calculated from the linear portion of the graphs,
as depicted in Figure 5, in the time intervals after the
first two minutes.

Within the first two minutes after starting these
reactions by the addition of either enzyme extract or
substrate (G-3-P), an extremely rapid increase in O.D. was
observed in all cases. This initial increase was immed-
iately followed by a decreased rate which followed a linear
pattern as depicted in Figures 5 and 6. In Figure 6,
it is shown that this rapid increase can be obtained
upon the addition of another aliquot of enzyme extract.
This step-like pattern can be repeated numerous times by
the addition of aliquots of enzyme extract. The same
pattern was obtained with either buffer or 2,4-D treated
enzyme extracts. The rapid increase in O.D. immediately
following the addition of an aliquot of enzyme extract
is the same following each addition of extract (0.065
per 30 seconds). The straight line portion of the curve
following the initial increase after the first addition
of an aliquot of enzyme extract is a change in O.D. of
0.0063 per 30 seconds, while following the second addition

63

TADLE 8

ACTIVITY OF GLYCERALDEIi/DE-3-PHOSPrIATE DEWDROGEInIASE^

Experiment Treatment"^*^ Change in O.D. per % Decrease
Number mg N per 5 min. Due to 2,4-D

23 Buffer . 0.48

2,4-D 0.42 13

24 Buffer 0.56

2,4-D 0.45 20

25 Buffer 0.61

2,4-D 0.54 12

"^The reaction mixture contained: Tris buffer, pH 8.5.
150 U..1; DPN, 800 ug; sodium arsenate, 170 uM; potassium fiouride,
10 u./i; reduced glutathione, 15 u 1; glyceraldehyde-3-phosphate,
2000 ug; water to 3.0 ml total volume; and acetone powder
extract. Blanks omitted DP.J. Enzyme extracts were added to start
the reaction and the increase in O.D. at 340 mu was followed at
timed intervals.

■^*^See Table 1.

64

1

1 1

-\

2,4-D

1

-

V §

o

CM

-

/buffer

1 1

-I

UJ

-

If)

1

1 1 1 1 \

o

o
in

o o o o
o In o tfi
(M ~ —

N 6uj|o/9NiaV3a il3nM

UJ

<f o

I

•H
M

O.
X

a>u-i
x:

c

>

cn

(0

e
o

a>
0.-0

c-o

•H to

M (J U

(TJ 'H

X

M

4) 3

M X <U

<4-< "-< O

0) I S o

cr> c rH c Oi

•H (T3 OJ O

s

8

a,

01

c

•H

3

0)
M

3

d)
H

o
u

03

c

' 1 1

H

o

\

<

1-
X

lO

\

111

E
b

■\

»

\

Q
UJ

Q

1

o

CVJ

X.

Q

H

c

■

<

EXTRAC

1

E
2

\

ED O.lml.

O

1 1 1

V%

in

<D m t

lO t\J

(nuJOfrOAilSNSG

nvoiido

65

of an aliquot of enzyme extract, a change in O.D, of
0.0094 per 30 seconds was obtained (Figure 6).

The rapid increase in O.D. immediately following the
addition of an aliquot of extract or the addition of sub-
strate may be explained on the basis of a rapid reduction
of GPDM bound Dp I. GPDII has been shown by several workers,
with the enzyme from mammalian and yeast tissues (146,153,
154), to combine with DP:J in a ratio of about 2 moles of
DPM per mole of enzyme. Apparently both DPMM and DPM are
bound by the enzyme and both have different dissociation
constants. Stockwell (146) states that DPUW has about
one tenth of the affinity of DP:i for binding sites on the
enzyme. Other work suggests that DP; J is strongly
bound. Thus the sloiver linear rate after about two minutes
of reaction time is probably controlled by the rate of
dissociation of DPnil. These slower linear rates are not
additive with increased aliquots of extracts. This may be
explained on the basis of a build-up of DPiJH, which competes
with DP'A for sites on the enzyme. Other components of the
reaction mixture, i.e. substrate, phosphate, arseno-3-
phosphoglyceric acid or 3-PGA also may be bound, and thus
affect the rate of the reaction.

Phogphoglyceric kinase. The activity of phospho-
glyceric kinase vi/as studied by trapping 1,3-DPGA with
hydroxylamine. Phosphoglyceric kinase was demonstrated in
cell-free extracts from both buffer and 2,4-D treated corn
seedlings. Figure 7 gives the type of results obtained

66

in these experiments. Obviously, 2,4-D treatment of corn
seedlings did not affect the in vitro activity of phospho-
glyceric kinase.

Carboxylases . I^Io carboxylase activity could be
demonstrated in either cell-free extracts or acetone powder
extracts. Using pyruvate as the substrate, no CO2 was
evolved in the tv;o hour incubation period. This infor-
mation allowed an assay for the activity of phosphoglyceric
mutase, enolase and pyruvic kinase by measuring pyruvate,

PhQgphgqly eerie rnut^se. This enzyme was demonstrated
qualitatively in both acetone powder extracts and cell-free
extracts, but none of these experiments were successful in
quantitatively demonstrating the presence of phosphoglyceric
mutase. No differences were observed in the activities of
phosphoglyceric mutase in extracts from either buffer or
2,4-D treated corn seedlings. The results of these experi-
ments were very erratic; therefore, no conclusions will be
drawn from them.

Several factors probably contributed to the failure
to demonstrate phosphoglyceric mutase quantitatively. One
obvious limitation was the use of simple extracts without
attempting to purify the enzyme. Second, the product of the
enzyme was not measured, but rather pyruvate was measured,
which was formed by two additional reactions from 2-PGA.
Third, the two additional reactions also were catalyzed
by endogenous enzymes, thus one or both of these enzymes
could control the entire chain of reactions.

67

Enolase. In the experiments designed to study the
activity of pyruvic kinase, phosphatase activity in the
enzyme preparations was high. Phosphatase catalyzes the
conversion of PEP to pyruvate. Therefore, pyruvate v;as the
compound which was measured to determine the activity of
enolase. Enolase was active in extracts from both buffer
and 2,4-D treated tissue. A pyruvate standard was not made;
therefore, for comparison, Klett readings per mg protein
added are presented in Table 9.

The rate of enolase activity was not affected by
2,4-D treatment as indicated in Table 9. Identical Klett
readings were obtained from both 2,4-D and buffer treated
extracts following the ten-minute incubation period.
Similar results, not presented, also were obtained using
acetone powder extracts.

Py?ljy^C- kJ-Pgsg « McCollum et al. (105,106) reported
that pyruvic kinase activity in cell-free extracts from
corn seedlings was labile and lost by dialysis, but they
successfully demonstrated the enzyme when they used
extracts from acetone powders which were not dialyzed.
Similar lability of the enzyme was noted in these experi-
ments .

In these studies with acetone powder extracts, a high
endogenous phosphatase activity was noted in the reaction
mixtures minus ADP which made measurement of this enzyme
difficult. Inclusion of ADP in the reaction mixture did
not increase the pyruvate formation above the endogenous

68

ta;.le 9

ACTIVITY OF EliOlASE^

Experime

Number

•nt

Treatment**

Klett readings
per mg protein

26

Buffer
2,4-D

20
20

27

Buffer
2,4-D

36
37

28

Buffer
2,4-D

27
26

*The reaction mixture contained: Tris
buffer, pil 7.4, 150 u-l; 2-PGA, 4500 ug; ..IgCl

20 uM; up to 0.4 ml of cell-free extract; and
water to 3.0 ml total volume.

**See Table 1.

69

except in a few cases. V/hen the addition of ADP increased
the formation of pyruvate, the increase did not in any
case exceed the endogenous in the buffer treated extracts
over 2b percent and the 2,4-D treated extracts over eight
percent. Thus, buffer extracts indicated a higher pyruvic
kinase activity than 2,4-D extracts. Attempts to quanti-
tatively measure the rate of pyruvate formation in both
types of extracts were not successful. This may be ex-
plained on the basis of the lack of purity of the enzyme
extracts, enzyme lability and the high endogenous phos-
phatase activity.

These ifl Yii£2 studies of the enzymes of the glycoly-
tic pathway indicate that 2,4-D treatment of etiolated
corn seedlings decreases the activity of 6-phospho-
fructokinase, aldolase and glyceraldehyde-3-phosphate
dehydrogenase. The activity of pyruvic kinase, although
not quantitatively measured, also was slightly decreased
following 2,4-D treatment. At the same time, the activi-
ties of phosphoglucoisomerase, phosphoglyceric kinase
and enolase were similar in extracts from both buffer and
2,4-D treated tissue. Studies of phosphoglyceric mutase
were inconclusive.

DISCUSSION

In a study of the in yllrg activity of enzymes,
several inherent problems must be recognized. These
problems are concerned with definite factors which can
influence the observed results; therefore, they should
at least be stated and kept in mind when the results
are discussed and interpreted.

Several assumptions were made in these studies
which should be noted. First, the assumption was made
that 2,4-D did not affect the extraction of an enzyme
or enzymes from the treated tissue. V/hen similar
extraction procedures are followed with both treated
and untreated tissues, are similar percentages of each
enzyme extracted from both tissues? Second, in irj yitJ;**?
work, changes could occur in an enzyme or enzymes as a
result of the extraction procedure. .Jote that in the
results, the activities of several enzymes from treated
and untreated tissues were the same. One interpretation
of this might be that the completeness of extraction
was the same from both treated and untreated tissues.
Third, by the nature of this study it was not possible
to purify the enzymes; therefore side reactions and
substrate specificity of the enzymes were inherent

70

71

problems. A fourth problem is metabolic toxicity or
stimulation. The effects of non-physiological substances
which are often employed in studying a particular enzyme
such as sodium arsenate used in the study of glyceralde-
hyde-3-phosphate dehydrogenase, is a fifth problem. Sixth,
when a specific enzyme was studied in these experiments,
the reaction mixture was prepared according to a general
procedure which has been worked out for a reasonably pure
enzyme. Such factors as temperature, pM and co-factors of
the reaction have been determined for the pure enzyme, and
it was assumed that these were the required factors for the
enzymes in this study, although they were not purified and
were from another source. Some other factors which may
affect the results are presented later.

As noted in the review of literature, several workers
have reported that 2,4-D affects the level of protein-
nitrogen in treated tissues. If 2,4-D has this effect,
obviously, in a study of this type in which the results
are given on the basis of per mg M or per mg protein, this
could skew the results. In 50 consecutive 'A determinations,
2,4-D treated tissues had an increased 'J content in 20
determinations. In 16 determinations, buffer treated
tissues had the highest rj, and in 14 determinations, they
were equal. The N content varied as much as 20 percent in
a few instances, but in no instance did the J determinations
affect the trend of the observed results of an experiment.

72

In this study, if the seedlings were placed in deionized
water and soaked for periods above four hours, beginning after
the 12-hour treatment of the corn seedlings, the effects of
2,4-D on glucose-6-phosphate dehydrogenase and glyceralde-
hyde-3-phosphate dehydrogenase could be partially reversed.

Apparently vyhen the seedlings were soaked in deionized
water, substances or substance were exuded into the soaking
solution. Root extracts made following soaking from both
buffer and 2,4-D treated seedlings demonstrated similar
enzymatic activity. The deionized water used to soak the
seedlings was concentrated to about one ml. When aliquots
of this concentrated soaking solution were added to the
reaction mixtures containing the enzyme extracts, a slight
inhibition of enzyme activity was noted. In one experiment,
the increase in 2,4-D extracts made prior to soaking was
20.3 percent. Following soaking, the increase was only
4.2 percent. V/hen the concentrated soaking solution was
added to the reaction mixtures, the increase was 8.0 percent.
Several research workers have reported the exudation of
substances from roots which affected growth. Eliasson (39)
reported that wheat roots exuded a substance which was
inhibitory to the growth of wheat roots. He further noted
that washing roots of pea seedlings treated with 2,4-D
counteracted the inhibition of root elongation and swelling
caused by 2,4-D (38). He concluded that his results
indicated that a leakable, growth-inhibitory substance is
formed in the roots in the presence of 2,4-D. Fries and

73

Foreman (53) identified some amino acids and nucleic acid
derivatives in pea root exudates. Howell (76) also re-
ported an inhibitory substance in pea roots. Housley et
al. (75) extracted several grov;th- inhibiting and growth-
promoting substances from the roots of maize. Soybean
seedlings, particularly seedlings treated with 2,4-D,
contain a compound which acts as an inhibitor of oxidative
phosphorylation (90).

Consideration of this reversal of the effects of
2,4-D by soaking the treated seedlings raises a question
concerning another portion of this study. Since soaking
intact seedlings will reverse 2,4-D effects, why doesn't
dialysis of the extracts have similar effects? In this
connection it should be noted that glyceraldehyde-3-
phosphate dehydrogenase was studied in acetone powder
extracts which were not dialyzed. Thus, the reversal of
the effects of 2,4-D by soaking intact tissue ivas noted
on both dialyzed cell-free extracts and undialyzed acetone
powder extracts.

The results of this study support the observations by
Humphreys and Dugger (80,81,82) concerning the catabolism
of glucose in intact corn roots. Their in vivg work
suggested that 2,4-D treatment of etiolated corn seedlings
affected glucose catabolism through an increase in the
amount of glucose catabolized via the pentose phosphate
pathway. This ijQ yi;^ro study of the enzyme activity of
both the pentose phosphate pathway and the glycolytic

74

pathway Indicates that 2,4-D treatment enhances the activity
of the pentose phosphate pathway enzymes. At the same time,
the activities of some glycolytic enzymes v^ere inhibited
following 2,4-D treatment.

The results of these studies clearly raise the question
of whether or not the observed changes in enzyme activity
are of sufficient magnitude to affect the pathways of glu-
cose metabolism in intact tissues. Since both of the major
catabolic pathways of glucose were demonstrated in both
buffer and 2,4-D treated tissues, why should the pentose
phosphate pathivay be favored over the glycolytic pathway?
It is difficult to see why such small changes can affect
the metabolism of a plant. iJote should be taken of the
fact that glycolytic enzymes v/ere more difficult to demon-
strate and that acetone powder had to be used in some cases,
i.e. glyceraldehyde-3-phosphate dehydrogenase.

Two groups of research workers recently reported on
regulatory mechanism of carbohydrate metabolism. Racker
and co-workers (57,58,169,170,171) have worked extensively
on the regulatory mechanisms in carbohydrate metabolism via
the glycolytic pathway in reconstructed systems, in ascites
tumor cells and HeLa cells. Chance and Fiess (24,25,26,27,
28,29,72) also have studied metabolic control mechanisms
using ascite tumor cells and bakers' yeast cells.

V/u and Racker (170) found that enzymes are present in
ascites tumor cell extracts in sufficient quantities to
account for glucose utilization via glycolysis in intact

75

cells. Under certain conditions, they noted that
glycolysis was inhibited even though sufficient quantities
of enzymes were present. They postulated that some co-
factor or co-factors may be limiting the glycolytic path-
way. They demonstrated that the intracellular concentration
of inorganic phosphate fluctuated parallel to the rate of
glycolysis. Thus, they concluded that the concentration of
inorganic phosphate was a major limiting factor in glycoly-
sis .

In this series of experiments, the enzymes of both
glycolysis and the pentose phosphate pathway were present
in the extracts. Even though previous in vivQ work in-
dicated that 2,4-D treatment caused a shift of glucose
catabolism to the pentose phosphate pathway, and this work
supports this as indicated by the stimulation of the
pentose phosphate pathway and the inhibition of glycolysis,
it is difficult to explain the action of 2,4-D on the basis
of the enzyme activity of the two pathways. Since the
enzymes of both pathways are present, it is proposed that
a co-factor or co-factors may be limiting glycolysis, or
that the co-factors may be stimulating the pentose phos-
phate pathway. Thus, the actual presence of an enzyme may
not mean that the enzyme is active, but rather, that if the
proper co-factors are present, the enzyme is active.

Two major types of co-factors are affected by 2,4-D.
First 2,4-D affects the ion concentration; and second,
it indirectly affects nucleotides in various fashions.

76

Inorganic phosphate is a well-known co-factor of
glycolysis. Racker and co-workers (57,58,169,170,171)
demonstrated that inorganic phosphate may be a limiting
co-factor, and thus exert some metabolic control. Theorell
(150) demonstrated in 1935, that inorganic phosphate
inhibited glucose-6-phosphate dehydrogenase. Kravitz
and Guarino (95), in 1958, using an enzyme extract from
ascites tumor cells, also demonstrated that inorganic
phosphate inhibited the activity of glucose-6-phosphate
dehydrogenase. These observations could be interpreted
to indicate that with high inorganic phosphate content,
glycolysis is stimulated and the pentose phosphate pathway
is inhibited, while with low inorganic phosphate concentra-
tions, the pentose phosphate pathway is stimulated. Thus,
experimental results, such as those of Ormrod (118), in
which the inorganic phosphate dropped sharply within
five minutes, could indicate a shift to the pentose phos-
phate pathway. 2,4-D possibly affects other ions, but
only inorganic phosphate has been shown experimentally to
affect the metabolic control mechanisms.

Kravitz and Guarino (95) found that the pentose
phosphate pathway could be stimulated by additions of
TP.J or of electron acceptors for reduced TP.J. So any
action whereby TPrJ is formed, either by synthesis or by
oxidation of reduced TPW, may stimulate the pentose
phosphate pathway. Action of growth regulators through
nucleic acid metabolism was proposed and somewhat

77

w
ma

substantiated by Skoog (136,139,140). Key et al. (92),
orking with 2,4-D treated soybeans, concluded that 2,4-D
y affect the metaboliSTi of nucleotides which are involved
in mitochondria gravth. In certain 2,4-D treated tissue,
the mitochondria were swollen and their acid-soluble nucleo-
tide content increased. They concluded that growth induced
by auxins involves a growth of mitochondria, and they postu-
lated that this growth is regulated through nucleotide meta-
bolisa. Stimulation of the pentose phosphate pathway by
2,4-D can be correlated with the synthesis of nucleotides.
In studies on ribose metabolism, Hiatt and Lareau (73) re-
cently demonstrated that labeled glucose fed to tissues
hich were oxidizing glucose via the pentose phosphate path-
ay resulted in the synthesis of labeled ribose and of
nucleotides in which the sugar moiety was labeled similarly.
Thus, the increased utilization of pentose in these experi-
ments could result in an increased nucleotide synthesis
which could affect the metabolic control mechanism. Sie et
al, (135) recently described a system whereby sedoheptulose
monophosphate is formed from purine and pyrimidine nucleo-
tides. If these reactions are assumed to occur in plant
tissues and to be reversible, then the increased formation
of sedoheptulose in 2,4-D treated extracts, which was ob-
served in these experiments, could be related to nucleotide
metabolism, and thus to a metabolic control mechanism.

w
w

78

Thus, experimental data support the theory that
2,4-D could affect the metabolic control mechanisms. The
elucidation of this mechanism will be dependent upon further
research employing such research techniques as those of
Chance and Hess (30), whereby small differences can be
measured accurately in short-time intervals employing
intact tissues.

SUA.1ARY AND COrCLUSIONS

A series of experiments was conducted to study the
effects of 2,4-D on the in yii£2 activity of enzymes
extracted from etiolated corn roots in an attempt to
determine the mechanism whereby 2,4-D shifted the normal
pathway of glucose catabolism. Comparisons were made
between the activities of enzymes extracted from the
roots of both buffer and 2,4-D treated etiolated corn
seedlings .

Three-day-old etiolated corn seedlings were divided

-2
into two groups and treated either v;ith 10 h phosphate

buffer (pli 5.3), or with buffer plus 10""^ 4 2,4-D for
12 hours at 22°C. After the 12- hour treatment period,
the seedlings ivere removed, washed, blotted dry and the
roots excised. The roots were weighed and used to pre-
pare enzyme extracts. Cell-free extracts and acetone
powder extracts were prepared from each group of seed-
lings. The activities of the enzymes of both the gly-
colytic and the pentose phosphate pathway were studied?
employing either cell-free extracts or acetone pov/der
extracts as the enzyme source. Individual enzymes were
studied in reaction mixtures containing excess substrate
plus the known cof actors, with enzyme concentration being

79

80

the limiting factor of the reaction. The activity of each
enzyme was determined in extracts from both buffer and
2,4-D treated tissues.

The results of these studies justify the following
conclusions:

1. Glycolytic enzymes and the enzymes of the
pentose phosphate pathway were present in extracts from
roots of both untreated and 2,4-D treated corn seedlings.

2. The activities of 6-phosphofructokinase, aldolase
and glyceraldehyde-3-phosphate dehydrogenase were de-
creased in extracts from 2,4-D treated tissue, while the
activities of phosphoglucoisomerase, phosphoglyceric
kinase and enolase were not affected. Studies of phospho-
glyceric mutase were not conclusive,

3. The activities of glucose-6-phosphate dehydro-
genase and of 6-phosphogluconate dehydrogenase were
enhanced in extracts from 2,4-D treated tissue.

4. In extracts from 2,4-D treated tissue, there
was an increased utilization of R-5-P and an increased
formation of heptulose and hexose.

5. These i,n vi,1;ro studies support the in yiyo
observation that 2,4-D treatment of etiolated corn
seedlings affects glucose catabolism through an increase
in the amount of glucose catabolized via the pentose
phosphate pathway.

81

6, A theory is presented and discussed which
proposes that 2,4-D in some manner effects the normal
metabolic control mechanisms of intact cells.

BILiLIOGRAPlf/

1. Adamson, D. and 'A. Adamson. Auxin action on
coleoptiles in the presence of nitrogen and at
low temperature. Science 128:532-533. 1958.

2. Akers, T. J. and S. C. Fang. Studies in plant
metabolis-n. VI. Effect of 2.4-D on the meta-
bolism of aspartic acid and glutamic acid in the
bean plant. Plant Physiol. 31:34-37. 1956.

3. Armarego, W. L. F., 1. J. Canny and S. F. Cox.
.'Vletal-chelating properties of plant-growth sub-
stances. ;Jature 183:1176-1177. 1959.

4. Asana, l\. D., G. Verma and V. S. lani. _ Some
observations on the influence of 2,4-dichloro-
phenoxyacetic acid (2,4-D) on the growth and
development of two varieties of wheat. Physiol.
Plant. 3:334-352. 1950.

5. Ashwell, G. Cysteine-H2S04 reaction for hexose.
In . Methods in Lnzymology HI. S, P. Colowick
and !J. 0. Kaplan, Id. pp. 81-82. :;ew"i0rk:
Academic Press. 1957.

6. Avery, G. S., Jr. Stimulation of respiration
in relation to growth. In Plant Growth Sub-
stances. F. Skoog, Fd. pp. 105-109. -ladison,
Wisconsin: University of Wisconsin Press. 1951.

7. Axelrod, l. and R. S. ;andurski. Phosphoglyceryl
kinase in higher plants. J. ^-iol. Chem. 204:
939-948. 1953.

8. Bail, ■\. Die Diagnose der Pentosurie. Deut.
med. V/och. 28:253-254. 1902.

9. L'ass, S. T.. C. L. Hammer and M. M. Sell. Effects
of 2,4-dichiorophenoxyacetic acid on the mineral
contents of cranberry bean plants (Phaseolus
yulqaris) . Uch. State Univ. Agr. Fxp. Sta. Quart
Bull. 42:43-46. 1959.

82

83

10. Beck, W. S. Determination of triose phosphate and
proposed modifications in the aldolase method of
Sibley and Lehninger. J. Biol. Chem. 212:847-857.
1955.

11. Bennet-Clark, T. A. Salt accumulation and mode of
action of auxin. In Chemistry and Mode of Action
of Plant Growth Substances. R. L. Wain and F.
VVightman, Ed. pp. 284-291. New York: Academic
Press. 1956.

12. Berg. R. T. and L. W. McElroy. Effect of 2,4-D
on the nitrate content of forage crops and weeds.
Can. J. Agric. Sci. 33:354-358. 1953.

13. Berger, J. and G. S. Avery, Jr. Glutamic and
isocitric acid dehydrogenases in the Ayena coleop-
tile and the effect of auxins on these enzymes.
Amer. J. Bot. 31:11-19. 1944.

14. Berger, J. and G. S. Avery, Jr. The mechanism of
auxin action. Science 98:454-455. 1943.

15. Biswas, B. ;: . and S. P. Sen. Relationship betv/een
auxins and nucleic acid synthesis in coleoptile
tissues. Nature 183:1824-1825. 1959.

16. Bonner, J., R. S. Bandurski and A. Millerd. Link-
age of respiration to auxin induced water absorp-
tion. Physiol. Plant. 6:511. 1953.

17. Boroughs, li. and J. Bonner. Effects of indoleacetic
acid on metabolic pathv;ays . Arch. Biochem. Biophys.
46:279-290. 1953.

18. Brakke, A. K. and L. G. r^chell. Lack of effect
of plant growth-regulators on the action of alpha-
amylase secreted by virus tumor tissue. Bot. Gaz.
113:482-484. 1952.

19. Brody, T. :A. Effect of certain plant growth sub-
stances on oxidative phosphorylation in rat liver
mitochondria. Proc. Soc. Exptl. Biol. Med. 80:
533-536. 1952.

20. Brown, J. v;. Effect of 2,4-D on the lUO relations,
the accumulation and distribution of solid matter,
and the respiration of bean plants. Bot. Gaz.
107:332-343. 1946.

21. Bryan, '.V. II. and E. II, Newcomb. Stimulation of
pectin methylesterase activity of cultured tobacco
pith by indoleacetic acid. Physiol. Plant. 7:290.
1954.

84

22. Burstrom, H. Studies on growth and metabolism of
roots. V. Cell elongation and dry matter content.
Physiol. Plant. 4:199-207. 1951.

23. Carr, D. J. and F. K. Ag, Residual effects of auxin,
chelating agents, and metabolic inhibitors in cell
extension. Aust. J. 3iol. Sci. 12:373-387. 1959.

24. Chance, B. Phosphorylation efficiency of the intact
cell. II. Crossover phenomena in aker's yeast.

J. uiol. Chem. 234:3036-3040. 1959.

25. Chance, E. Phosphorylation efficiency of the intact
cell. III. Phosphorylation and dephosphorylation

in yeast cells. J. liiol. Chem. 234:3041-3043. 1959.

26. Chance, 3. and B. !-Iess. Metabolic control mechanisms.

I. Electron transfer in the mammalian cell. J. Eiol.
Chem. 234:2404-2412. 1959.

27. Chance, B. and B. Hess. Metabolic control mechanisms.

II. Crossover phenomena in mitochondria of ascites
tumor cells. J. -iol. Chem. 234:2413-2415. 1959.

28. Chance. 3. and B. Hess. Metabolic control mechanisms.

III. Kinetics of oxygen utilization in ascites tumor
cells. J. ^iol. Chem. 234:2416-2420. 1959.

29. Chance, 3. and B, Hess. letabolic control mechanisms.

IV. The effect of glucose upon the steady state
respiratory enzymes in the ascites cell. J. Biol.
Chem. 234:2421-2427. 1959.

30. Chance, 3. and 3. Hess. Spectroscopic evidence of
metabolic control. Science 129:700-708. 1959.

31. Clayton, R. A. Pentose cycle activity in cell-free
extracts of tobacco leaves and seedlings. Arch.
Biochem. Biophys. 79:111-123. 1959.

32. Cohen, D.. ^. Ginsburg and C. Heitner-Wirguin.
i4etal-cheiating properties of plant-growth substances.
Nature 181:686-687. 1958.

33. Cooke, A. R. Influence of 2,4-D on the uptake of
minerals from the soil. Weeds 5:25-28. 1957.

34. Corns, W. G. Effects of 2,4-D and soil moisture on
the catalase activity, respiration and protein con-
tent of bean plants. Cana. J. Res. 28C: 393-405. 1950.

35. Croker, 3. H. Effects of 2,4-dichlorophenoxyacetic
acid and 2,4,5-trichlorophenoxyacetic acid on mitosis
in Allium cepa. Bot. Gaz. 114:274-283. 1953.

85

36. Datta, S. C. and S. Dunn. Effects of light qual-
ity on herbicide toxicity to plants. iVeeds 7:55-65.
1959.

37. Dische, Z., L. 3. Shettles and M. Osnos. Nev; specif-
ic color reactions of hexoses and spectrophotometric
micromethods for their determination. Arch. Biochem.
22:169-184. 1949.

38. Eliasson, L. Growth response of pea roots to 2,4-D
applied to the hypocotyl. Physiol. Plant. 12:834-
840. 1959.

39. Eliasson, L. Inhibition of the growth of wheat
roots in nutrient solutions by substances exuded
from the roots. Ann. Royal Agr. Coll. Sweden.
25:285-293. 1959.

40. Erickson, L. C., C. I. Seeley and K. H. Klages.
Effect of 2,4-D upon the protein content of wheats.
J. Amer. Soc. Agron. 40:659-660. 1948.

41. Erickson, L. C, R. T. Wedding and h , L. 2rannaman.
Influence of pii on 2,4-dichlorophenoxyacetic acid

and acetic acid activity in Qii£££Jj,^ Plant Physiol.
30:69-74. 1955.

42. Fang, S. C., F. Tenny and J. S. Butts. Influence of
2,4-dichlorophenoxyacetic acid on pathv/ays of glu-
cose utilization in bean stem tissues. Plant Physiol.
35:405-408. 1960.

43. Fang, S. C. and J. S. Butts. Studies in plant meta-
bolism. IV. Comparative effects of 2,4-dichloro-
phenoxyacetic acid and other plant growth regulators

on phosphorus metabolism in bean plants. Plant Physiol.
29:365-368. 1954.

44. Fav;cett, C. H. Plant-growth substances and the
copper chelation theory of their mode of action.
Nature 184:796-798. 1959.

45. Fischer, E. H. and J. Fellig. Salivary amylase in-
hibition. Science 115:684-685. 1952.

46. Frank, P. A. and 3. !I. Grigsby. Effects of herbicidal
sprays on nitrate accumulation in certain weed species.
Weeds 5:206-217. 1957.

47. Freeland, R. O. Effects of growth substances on
photosynthesis. Plant Physiol. 24:621-628. 1949.

86

48. Freiberg, S, R. Effects of exogenous growth
regulator on proteolytic enzymes of the soybean
plant. Science 115:674-675. 1952.

49. Freiberg, S. R. and H. E. Clark. Changes in
nitrogen fractions and proteolytic enzymes of
soybean plants treated with 2,4-dichlorophenoxy-
acetic acid. Plant Physiol. 30:39-46. 1955.

50. Freiberg, S. R. and H. E. Clark. Effects of
2,4-dichlorophenoxyacetic acid upon the nitrogen
metabolism and water relations of soybean plants
grown at different nitrogen levels. n.ot. Gaz.
113:322-333. 1952.

51. French, R. C. and H. Beevers. Respiratory and
growth responses induced by growth regulators and
allied compounds. Amer. J. 3ot . 40:660-666. 1953.

52. Friedemann, T. F. and G. E. Maugen. Pyruvic acid.
II. The determination of keto acids in blood and
urine. J. Biol. Chem. 147:415-442. 1943.

53. Fries, N. and B. Forsman. Quantitative determina-
tion of certain nucleic acid derivatives in pea
root exudates. Physiol. Plant. 4:410-420. 1951.

54. Fults, J. L. and M. G. Payne. Effects of 2,4-
dichlorophenoxyacetic acid and maleic hydrazide on
free amino acids and proteins in potato, sugar
beet, and bean tops. l:ot. Gaz. 118:130-133. 1956.

55. Galston, A. W. and R. Kaur. An effect of auxins on
the heat coagulability of the proteins of growing
plant cells. Proc. Natl. Acad. Sci. 45:1587-1590.
1959.

56. Galston, A. v;. and W. K. Purves . The mechanism of
action of auxin. Ann. Rev. Plant Physiol. 11:239-
276. 1960.

57. Gatt, S. and E. Racker. Regulatory mechanisms in
carbohydrate metabolism. I. Crabtree effect in
reconstructed systems. J. lIoI. Chem. 234:1015-
1023. 1959.

58. Gatt, S. and E. Racker. Regulatory mechanisms in
carbohydrate metabolism. H. Pasteur effect in
reconstructed systems. J. ^iol. Chem. 234:1024-
1028. 1959.

87

59. Gibbs, '1. Triosephosphate dehydrogenase and glucose-
6-phosphate dehydrogenase in the pea plant. :Wature
170:164. 1952.

60. Glaszious, K. T. The effect of auxins on the binding
of pectin methylesterase to cell wall preparations.
Aust. J. liol. Sci. 10:426-434. 1957.

61. Glasziou, K. T. Effect of 2,4-dichlorophenoxyacetic
acid on pectin methylesterase in tobacco pith sections.
Nature 181:428-429. 1958.

62. Glaszious, K. T, and S. D. Inglis. The effect of
auxin on the binding of pectin methylesterase to
cell walls. Aust. J. Biol. Sci. 11:127-141. 1958.

63. Gordon, S. A. Auxin-protein complexes of the wheat
grain. Amer. J. Bot . 33:160-169. 1946.

64. Grisolia, S., L. C. lokrasch and V. D. Hospelhorn.
Adenosine mono-, di- and triphosphate, pyruvic kinase,
hexokinase and polynucleotide phosphorylase assay.
Biochem. Biophys. Acta. 28:350-354. 1958.

65. Hageman, R. H. and D. I. Arnon. Changes in glycer-
aldehyde phosphate dehydrogenase during the life
cycle of a green plant. Arch. Biochem. and Biophys.
57:421-436. 1955.

66. liagen^ C. E., C, 0. Clagett and E. A. Helgeson.
2,4-dichlorophenoxyacetic acid inhibition of castor
bean lipase. Science 110:116-117. 1949.

67. Hanson, J. I- . and J. Bonner. The relationship between
salt and water uptake in Jersulem artichoke tuber
tissue. Amer. J. Bot. 41:702-710. 1954.

68. Heath, 0. V . S. and J. E. Clark. Chelating agents
as plant growth substances. A possible clue to
the mode of action on auxin. Hature 177:1118-1121.
1956.

69. iieath, 0. V. S. and J. E. Clark. Chelating agents
as growth substances. Hature 178:600-601. 1956.

70. Heath. 0. V. S. and J. E. Clark. Comment on the
article by Armarego. Mature 183:1177. 1959.

71. Henderson, J. 11. ..'.., I. H. Miller and D. C. Desse.
Effect of 2,4-dichlorophenoxyacetic acid on respiration
and on destruction of indoleacetic acid in oat and
sunflower tissues. Science 120: 710-712. 1954.

88

72. Hess, B. and B. Chance. Phosphorylation effeciency
of the intact cell. I. Glucose-oxygen titrations
in ascites tumor cells. J. Biol. Chem. 234:3031-
3055. 1959.

73. Hiatt, H. H. and J. Lareau. Studies of ribose meta-
bolism. VIII. Pathways of ribose biosynthesis in
vivo and in vitrp in rat, mouse, and human tissues.
J. Biol. Chem. 235:1241-1245. 1960.

74. Horecker, B. L. and P. Z. Smyroniotis. The enzymatic
formation of sedoheptulose phosphate from pentose
phosphate. J. Amer. Chem. Soc . 74:2123. 1952.

75. Housley, S., A. Booth and I. D. J. Phillips. Stimu-
lation of coleoptile and root-growth by extracts of
maize. iJature 178:255-256. 1956.

76. Howell, R. W. The inhibiting effect of root tips

on the elongation of excised Pisum epicotys cultures
in yii£2. Plant Physiol. 29:100. 1954.

77. Hsueh, Y. L. and C. H. Lou. Effect of 2,4-D on seed
germination and respiration. Science 105:283-285.
1947.

78. Humphreys, T. E. and W. ^. Dugger, Jr. The effect
of 2,4-dichlorophenoxyacetic acid on the respiration
of etiolated pea seedlings. Plant Physiol. 31
Suppl.:xxii. 1956.

79. Humphreys, T. E. and W. 1. Dugger, Jr. The effect
of 2,4-dichlorophenoxyacetic acid on pathways of
glucose catabolism in higher plants. Plant Physiol.
32:136-140. 1957.

80. Humphreys, T. E. and W. A. Dugger, Jr. The effect
of 2,4-dichlorophenoxYacetic acid on the respiration
of etiolated pea seedlings. Plant Physiol. 32:530-
536. 1957.

81. Humphreys, T. E. and W. .1. Dugger, Jr. Effect of
2,4-dichlorophenoxyacetic acid and 2,4-dinitrophenol
on the uptake and metabolism of exogenous substrates
by corn roots. Plant Physiol. 34:112-116. 1959.

82. Humphreys. T. E. and IV. A. Dugger, Jr. Use of
specifically labeled glucose and gluconate in the
evaluation of catabolic pathways for glucose in
corn roots. Plant Physiol. 34:580-582. 1959.

89

83. Johnson, E. J. and A. R. Colmer. Relationship
between magnesium and the physiological effects of
2,4-dichlorophenoxyacetic acid on Azotobacter
yinelandii and Rhizobium meliloti. Jour. Bact.
73:139-143. 1957.

84. Johnson, E. J. and A. R. Colmer. Further studies
on the mode of action of 2,4-dichlorophenoxyacetic
acid on Azotobacter vinel^ndii as related to mag-
nesium and phosphate. Jour. 3act. 73:666-669. 1957.

85. Johnson, E. J. and A. R. Colmer. The relation of
magnesium ion to the inhibition of the respiration
of Azo1;Qbacjey yioe^apdii by aureomycin, achromycin
and 2,4-dichlorophenoxyacetic acid. Antibiotics
and Chemotherapy 7:521-526. 1957.

86. Johnson, E. J. and A, R. Colmer. The relationship
of magnesium ion and molecular structure of 2,4-
dichlorophenoxyacetic acid and some related com-
pounds to the inhibition of the respiration of
AzQtpbacter vinejt^ndii. Plant Physiol. 33:99-101.
1958.

87. Kachmar, J. F. and P. D. Boyer. Kinetic analysis

of enzyme reactions. I. The potassium activation and
calcium inhibition of pyruvic phosphof erase. J,
fliol. Chem. 200:669-682. 1953.

88. Kelly, S. and G. S. Avery. The effect of 2,4-dichloro-
phenoxyacetic acid and other physiologically active
substances on respiration. Amer. J. I3ot. 36:421-426.
1949.

89. Kelly, S. and G. S. Avery. The age of pea tissue
and other factors influencing the respiratory
response to 2,4-dichlorophenoxyacetic acid and
dinitro compounds. Amer. J. Bot . 38:1-5. 1951.

90. Key, J. L. and D. S. Galitz. Growth inhibitor in
immature soybean seeds and 2,4-D sprayed seedlings.
Science 130:1339-1340. 1956.

91. Key, J. L. and J. B. Hanson. Accumulation of a
naturally occuring growth inhibitor in soybeans
following treatment with 2,4-D. Plant Physiol.
Suppl.:xvii. 1959.

92. Key, J. L., J. B. Hanson and R. F. Bils. Effect of
2,4-dichlorophenoxyacetic acid application on activi-
ty and composition of mitochondria from soybeans.
Plant Physiol. 35:177-183. 1960.

90

93. Klevstrand, R. and A. Nordal. A spraying reagent
for paper chromatograms which is apparently specific
for ketoheptoses. Acta. Chem. Scand. 4:1320. 1950,

94. Klingman, G. C. and G. H. Ahlgren. Effects of 2.4-D
on dry weight, reducing sugars, total sugars, poly-
saccharides, nitrogen and ally! sulfide in wild
garlic. 3ot. Gaz. 113:119-134. 1951.

95. Kravitz, E. A. and A. J. Guarino. On the effect of
inorganic phosphate on hexose phosphate metabolistn.
Science 128:1139-1140. 1958.

96. Krimsky, I. and E. Backer. Glutathione, a prosthetic
group of glyceraldehyde-3-phosphate dehydrogenase.

J. Biol. Chem. 198:721-729. 1952.

97. Kvamme, 0. J., C. 0. Clagett and W. B. Treumann.
Kinetics of the action of the sodium salt of 2,4-
dichlorophenoxyacetic acid on the germ lipase of wheat,
Arch. 3iochem. 24:321-328. 1949.

98. Ling, Kuo-Huang, VV, L. Byrne and H. Lardy. Phospho-
hexokinase. In Methods in Enzymology. I. S. P.
Colowick and r4. P. Kaplan, Ed. pp. 306-308.

New York: Academic Press. 1955.

99. Livingstone, C., :A. G. Payne and J. L. Fultz. Effects
of maleic hydrazide and 2,4-dichlorophenoxyacetic
acid on the free amino-acids in sugar beets. Bot.
Gaz. 116:148-156. 1954.

100. Loustalot, A. J. and T. J. Muzik. Effect of 2,4-D
on apparent photosynthesis and developmental mor-
phology of Velvet Bean. Bot. Gaz. 115:56-66. 1953.

101. Loustalot, A. J., 4. P. /lorris, J. Garcia and C.
Pagan. 2,4-D affects phosphorus metabolism. Science
118:627-628. 1953.

102. Luecke, R. W., C. L. Hammer and H. :A. Sell. Effect
of 2.4-dichlorophenoxyacetic acid on the content

of thiamine, riboflavin, nicotinic acid, pantothenic
acid and carotene in stems and leaves of red kidney
bean plants. Plant Physiol. 24:546-548. 1949.

103. Marcus, A. Photocontrol of formation of red kidney
bean leaf triphosphopyridine nucleotide linked
triosephosphate dehydrogenase. Plant Physiol.
35:126-128. 1960.

91

104. -larre, E. and G. Forti. Metabolic responses to
auxin. III. The effects of auxin on ATP level
as related to the auxin induced respiration in-
crease. Physiol. Plant. 11:36-47. 1958.

105. McCollum, R. E., R. H. Hageman and E. H. Tyner.
Influence of potassium on pyruvic kinase from
plant tissue. Soil Science 86:324-331. 1958.

106. 4cCollum, R. E., R. H. Hageman and E. H. Tyner.
Occurance of pyruvic kinase and phosphoenolpyru-
vate phosphatases in seeds of higher plants.
Soil Science 89:49-52. 1960.

107. -IcElroy, W. C. and 3. Glass, Ed. Symposium on ^
phosphorus metabolism. I. Baltimore: Johns
Hopkins Press. 1951.

108. .IcElroy, 'V. C. and '-^ . Glass, Ed. Symposium on
phosphorus metabolism. II. Baltimore: Johns
i^opkins Press. 1952.

• •

109. Aejbaum, W. Uber die 3estimmung Kleiner Pento-
semengen, insbesondere in Derivaten der Adenyl-
saiire. Z. Physiol. Chem. 258:117-120. 1939.

110. ..Uller, I. H. and R. H. Burris . Effect of plant
growth substances upon oxidation of ascorbic and
glycolic acids by cell-free enzymes from barley.
Amer. J. Bot . 38:547-549. 1951.

111. Mitchell, J. VI, and J. W. Brown. Effect of 2,4-D

acid in the readily available carbohydrate constituents
in annual morning-glory. Bot. Gaz. 107:120-129. 1945.

112. Mitchell, J. W., R. H. Burris and A. J. Riker.
Inhibition of respiration in plant tissues by
callus stimulating substances and related chemicals.
Amer. J. Bot. 36:368-377. 1949.

113. loewus, Franz. The action of 2,4-D on deaminating
enzymes. 8th International Bot. Congress, pp. 149-
150. 1954.

114. Nance, J. F. Inhibition of salt accumulation in
excised wheat roots by 2,4-dichlorophenoxyacetic
acid. Science 109:174-176. 1949.

115. Neely, W. B., C. D. Ball, C. L. Hamner and H. A.
Sell. Effect of 2,4-dichlorophenoxyacetic acid on
the alpha and beta amylase activity in the stems
and leaves of red kidney bean plants. Science 111:
118. 1950.

92

116. Nickell, L. G. Effect of certain plant hormones
and colchicine on the growth and respiration of
virus tumor tissue from Rurqex aceto^a. Amer. J.
Bot. 37:829-835. 1950.

117. Ordin, L., R. Cleland and J. Bonner. Methyl ester-
ification of cell wall constituents under the in-
fluence of auxin. Plant Physiol. 32:216-220. 1957.

118. Ormrod, D. P. and W. A. Williams. Phosphorus meta-
bolism of Trif olium hirtum All. as affected by
2,4-dichlorophenoxyacetic acid and gibberellic
acid. Plant Physiol. 35:81-87. 1960.

119. Payne, M. G., J. L. Fults and R. J. Hay. Amino
acids in potato tubers altered by 2,4-D treatment
of plants. Science 114:204-205. 1951.

120. Rakitin, Y. V. and A. K. Potapova. Penetration
into plants of herbicides and their influence on
phosphorus uptake. Fiziologiia Rastenii. 6:621-
623. 1960.

121. Rakitin, Y. V. and V. A. Zemskaia. The effect of
2,4-D on nitrogen metabolism in oat and bean plants.
Fiziologiia Rastenii. 5:190. 1958.

122. Rasch, E., H. Swift and R. 4. Klein. "Jucleopro-
tein changes in plant tumor growth. J. Biophysic.
and Biochem. Cytol. 6:11-34. 1959.

123. Rasmussen, L. W. The physiological action of 2,4-
dichlorophenoxyacetic acid in dandelion. Taraxacum
officinale. Plant Physiol. 22:377-392. I947.

124. Rebstock. T. L., C. L. Hamner, C. D. Ball and

H. A. Sell. Effect of 2,4-dichlorophenoxyacetic acid
on proteolytic activity of red kidney bean plants.
Plant Physiol. 27:639-643. 1952.

125. Rebstock, T. L., C. L. Hamner and H. M. Sell. The
influence of 2,4-dichlorophenoxyacetic acid and the
phosphorus metabolism of cranberry bean plants
(Phaseo],!j_s yulgari?) . Plant Physiol. 29:490-491.
1954.

126. Rhodes, A. The influence of the plant growth-
regulator, 2-methyl-4-chlorophenoxyacetic acid,
on the metabolism of carbohydrate, nitrogen and
minerals in Spl^num lycopersicum. J. Exp. ^ot.
3:129-154. 1952.

93

127. Rhodes, A., W. G. Templeman and M. N. Thruston.
The effect of plant growth regulator, 4-chloro-2-
methyl phenoxyacetic acid, on the mineral and
nitrogen content of plants. Ann. 3ot. (n.s.)
14:181-198. 1950.

128. Rice, E. L. and L. M. Rohrbaugh. Relation of
potassium nutrition to the translocation of 2,4-
dichlorophenoxyacetic acid in tomato plants.
Plant Physiol. 33:300-303. 1958.

129. Roe, J. H. A colorimetric method for the deter-
mination of fructose in blood and urine. J.
Biol. Chem. 107:15-22. 1934.

130. Rohrbaugh, L. .1. and E. L. Rice. Relations of
phosphorus nutrition to the translocation of 2,4-
dichiorophenoxyacetic acid in tomato plants. Plant
Physiol. 31:196-199. 1956.

131. Sell, H. VI., C. L. Hamner, T. L. Rebstock and

L. E. Weller. The effect of monochlorophenoxyacet ic
acids on the composition of bean plants (Pha?eolu^
vulqa^ris) . Quart. Cull, of the Mich. Agric. Exp.
Sta. 40:306-310. 1957.

132. Sell, H, M., R. \'l. Luecke, 3. M. Taylor and C. L.
Hamner. Changes in chemical composition of stems

of red kidney bean plants treated with 2,4-dichloro-

phenoxyacetic acid. Plant Physiol. 24:295-299.

1949.

<■

133. Shaw, W. C., C. J. VVilard and R. L. Bernard. The
effect of 2,4-dichlorophenoxyacetic acid (2,4-D)
on wheat, oats, barley and the legumes underseeded
in these crops. Ohio Agr. Exp. Sta. Res. 3ull. 761.
1955.

134. Sibley, J. A. and A. L. Lehninger. Determination
of aldolase in animal tissues. J. 3iol. Chem.
177:359-872. 1949.

135. Sie, Hsien-Giett, V. M. lUgam and VI. H. Fishman.
Conversion of nucleoside to sedoheptulose monophos-
phate by rat liver. J. Biol. Chem. 234:1202-1207.
1959.

136.

Silberger, J. and F. Skoog. lAA-induced changes in
nucleic acid contents and grov/th of tobacco pith
tissue. Science 118:443-444. 1953.

94

137. Singer. T. P. Plant Carboxylases. In .'Aethods of
Enzymoiogy. I. S. P. Colowick and M. O. Kaplan, Ed.
pp. 460-471. Mew York: Academic Press. 1957.

138. Singer, T. P. and J. Pensky. Isolation and properties
of the alpha-carboxylase of wheat germ. J. Biol.
Chem. 196:375-388. 1952.

139. Skoog, F. Substances involved in normal growth and
differentiation of plants. 3rookhaven Symposia in
Biology 6(B:JL 258): 1-21. 1954.

140. Skoog, F. and ;3. J. Robinson. A direct relation-
ship between indoleacetic acid effects on growth
and reducing sugars in tobacco tissue. Proc. Soc.
Exp. 3iol. 'led. 74:565-568. 1950.

141. Slein, M. W. Phosphoglucose Isomerase. In Methods

in Enzymoiogy. I. S. P. Colowick and J, 0. Kaplan, Ed.
pp. 304-306. .Jew York: Academic Press. 1955.

142. Smith, F. G. Effects of 2,4-D on the respiratory
metabolism of bean stem. Plant Physiol. 23:70-80.
1948.

143. Smith, F. G., C. L. Hamner and R. F. Carlson.
Changes in food reserves and respiratory capacity
of bindweed tissues accompanying herbicidal action
of 2,4-D. Plant Physiol. 22:58-65. 1947.

144. Smith, I. Ed. Chromatographic Techniques, 1st Ed.
Mew York: Interscience Publishers, Inc. 1958.

145. Stahler, L. .1. and E. I. Whitehead. The effect of
2,4-D on potassium nitrate levels in leaves of
sugar beets. Science 112:749-751. 1950.

146. Stockell, Anne. The binding of diphosphopyridine
nucleotide by glyceraldehyde-3-phosphate dehydrogen-
ase. J. oiol. Chem. 234:1286-1292. 1959.

147. Switzer, C. A. Effects of herbicides and
related chemicals on oxidation and phosphorylation
by isolated soybean mitochondria. Plant Physiol.
32:42-44. 1957.

148. Tagawa, T. and J. Bonner. Mechanical properties of
the Aven^i coleoptile as related to auxin and to
ionic interactions. Plant Physiol. 32:207-215. 1957.

149. Taylor, D. L. Effects of 2,4-D on gas exchange of
wheat and mustard seedlings. 3ot. Gaz. 109:162-176.
1948.

95

150. Theorell, H. Uber Hemmung der Reaktions-geschwind-
igkeit durch Phosphat in Warburgs und Christians
System. Biochem. Z. 275:416-421. 1935.

151. Tukey, H. B., C. L. 1-Iamner and !^. Moore. Histo-
logical changes in bindweed and sow thistle
following applications of 2.4-D in herbicidal
concentrations. Bot . Gaz. 107:62-73. 1945.

152. Umbreit, W. '.'.'., R. H. Burr is and J. F. Stauffer.
4anometric Techniques, 3rd Ed. pp. 28-45.
.Minneapolis: Burgess. 1957.

153. Velick, S. F. The alcohol and glyceraldehyde-3-
phosphate dehydrogenases of yeast and mammals. In
Mechanisms of Enzyme Action. V/. C. McElroy and B.

Glass, Ed. pp. 491-519. Baltimore: Johns Hopkins
Press. 1954.

154. Velick, S. F. Flouresence spectra and polariza-
tion of glyceraldehyde-3-phosphate and lactic
dehydrogenase co-enzyme complexes. J. Biol. Chem.
233:1455-1467. 1958.

155. Vennesland, •■'> . and E. Conn. Carboxylating
enzymes in plants. Ann. Rev. Plant Physiol. 3:
307-332. 1952.

156. Volker, J. F. Compounds capable of plant amylase
inhibition. Science 112:61. 1950.

157. Wagenknecht, A, C., A. J. Riker, T. C. Allen and
R. H. Burrss. Plant growth substances and the
activity of cell-free respiratory enzymes. Amer.
J. Bot. 38:550-554. 1951.

158. Warburg, 0, and 'V. Christian. Isolierung und
Kristaliisation des Proteins des oxydierenden
Gtfrunsferments. Biochem. Z. 303:40-68. 1939.

159. V/edding, R. T., L. C. Erickson and B. L. 'rannaman.
Effect of 2,4-D on photosynthesis and respiration.
Plant Physiol. 29:64-69. 1954.

160. Wedding, R. T., L. C. Erickson and M. Kay Black.
Influence of 2.4-dichlorophenoxyacetic acid on
solute uptake by Chlorella. Plant Physiol. 34:
3-10. 1959.

96

161. Weller, L. E., R. '.V. Leuke, C. L. Hamner and II. 'A.
Sell. Changes in chemical composition of the leaves
and roots of red kidney beans treated with 2,4-D.
Plant Physiol. 25:289-293. 1950.

162. West, F. R. Jr. and J. H. -1. Henderson. The effect
of 2,4-dichiorophenoxyacetic acid and various other
substances upon the respiration of blue lupine seed-
ling roots. Science 111:579-581. 1950.

163. West, S. H., J. B. Hanson and J. L. Key. Effect of
2,4-dichlorophenoxyacetic acid on the nucleic acid
and protein content of seedling tissue. Weeds

In Press.

164. Wilden, C. F., C. L. Hamner and S. T. Bass. The
effect of 2,4-dichlorophenoxyacetic acid on the
accumulation of mineral elements in tobacco plants.
Plant Physiol. 32:243-244. 1957.

165. Wolf, D. E., G. Vermillion, A. Wallace and G. H.
Ahlgren. Effect of 2,4-D on carbohydrate and
nutrient element content and on rapidity of kill

of soybean plants growing at different nitrogen levels.
Bot Gaz. 112:188-197. 1950.

166. Wort, D. J. Effects of non-lethal concentrations
of 2,4-D on buckwheat. Plant Physiol. 26:50-58.
1951.

167. Wort, D. J. The response of buckwheat to treatment
with 2,4-dichlorophenoxyacetic acid. Amer. J. 3ot.
36:673-676. 1949.

168. V;ort, D. J. and L. 'A. Cowie. The effect of 2,4-D
on phosphorylase, phosphatase, amylase, catalase
and peroxidase activity in v^heat. Plant Physiol.
28:135-139. 1953.

169. I'/u, R. Regulatory mechanisms in carbohydrate
metabolism. V. Limiting factors of glycolysis in
ileLa cells. J. Biol. Chem. 234:2806-2810. 1959.

170. iVu, R. and E. Racker. Regulatory mechanisms in
carbohydrate metabolism. III. Limiting factors in
glycolysis of ascites tumor cells. J. ^iol. Chem.
234:1029-1035. 1959.

171. Wu, R. and E. Racker. Regulatory mechanisms in
carbohydrate metabolism. IV. Pasteur effect and
Crabtree effect in ascites tumor cells. J. Biol.
Chem. 234:1036-1041. 1959.

97

172. Yasuda, G. K., M. G. Payne and J. L. Gults. Effect
of 2,4-dichlorophenoxyacetic acid and maleic hydra-
zide on potato proteins as shown by paper electro-
phoresis. Mature 176:1029-1030. 1956.

BIOGRAPi-IICAL SKETCH

Clanton C. Black, Jr., was born November 27, 1931,
in Tampa, Hillsborough County, Florida. He attended
elementary school at 4ango, Florida, until he was ten
years old. He completed his education at Gainesville,
Florida.

After graduation from Gainesville High School in
1949, he entered the University of Florida, where he
majored in Agronomy. During his third year of college,
he married Betty L. Dantzler. The Degree of Bachelor of
Science in Agriculture was conferred on him by this same
institution in August, 1953.

He returned to the University of Florida in Septem-
ber, 1955, to pursue graduate work in the field of Agron-
omy, specializing in Chemical Weed Control. The Degree
of Master of Science in Agriculture was conferred on him
in June, 1957.

In September, 1957, he began further graduate studies
at the University of Florida, majoring in Agronomy. In
August, 1960, the Degree of Doctor of Philosophy was con-
ferred on him.

He is a member of the following honorary societies:
Gamma Sigma Delta, Phi Sigma and Sigma Xi.

98

This dissertation was prepared under the direction
of the chairman of the candidate's supervisory committee
and has been approved by all members of that committee.
It was submitted to the Dean of the College of Agriculture
and to the Graduate Council, and was approved as partial
fulfillment of the requirements for the degree of Doctor
of Philosophy.

August, 1960

> ^ /^^t-CJ-TT^^

Dean, College of Agriculture

Supervisory Committee:
Chairman '?T

Dean, Graduate School

1 'y^