SOME EFFECTS OF
2,4-DICHLOROPHENOXYACETIC ACID ON
THE CARBOHYDRATE METABOLISM
OF ETIOLATED CORN SEEDLINGS
CLANTON C. BLACK, JR.
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
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. O. Spinks
and T. VI. Stearns, for service on the committee; and to the
Department of Agronomy for financial support during this
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.
Betty Louise Black
TA-.LE OF CONTENTS
ACKNOWLEDGEMENT . . . . . . . ... ii
DEDICATION . . . . . . . . . . iii
LIST OF TABLES . . . . .. . . .. vi
LIST OF FIGURES . . . . . . . . .. vii
STATEMENT OF PROLLE .. . . . . . . 1
REVIEW OF LITERATURE . . . . . . . 4
Effects of 2,4-D on Gas Exchange
Effects of 2,4-D on .ineral 'etabolism
Effects of 2,4-D on the Aetabolism of Carbon
Compounds and Related Enzyme Systems
4ATERIALS . . . . . . . . . . 24
Preparation of enzyme extracts
EXPERIMENTAL PROCEDURE . . . . . . . 28
Studies on the Pentose Phosphate Pathway
Chromatographic studies on pentose disappear-
Assays for specific enzymes
Studies on the Glycolytic Pathway
Phosphoglyceric mutase, enolase and pyruvic
Reagents used in these studies
RESULTS . . . . . . . . . 43
Studies on the Pentose Phosphate Pathway
Chromatographic studies on pentose disappear-
Studies on the Glycolytic Pathway
DISCUSSION . . . . . . . . . .70
SU.A.ARY A.ND COiJCLUSIONS . . . . . .. 79
I3 LIOGRAPHY .. .. . .. .... ... 82
LIST OF TABLES
1. PE:JTOSE UTILIZATION A'JD FCOR'TION OF
HEPTULOSE A:D IIEXOSE . . .... ... 44
2. DISTRIBUTION OF CARDO>4 ATO',S FROQ' RII'OSE-5-
PIOSPiiATE FOLLOW'I'IG 60- iI:UTE I;ICU.ATIO,'
PERIOD .. ... . . . . . 48
3. ACTIVITY OF GLUCOSE-6-PHOSPIATE DE:YDROGE!'-
ASE . . . . . . . . . 51
4. ACTIVITY OF 6-PI1OSPHIOGLUCO:1ATE DEHi'DREOGEIASE 54
b. ACTIVITY OF PIIOSPIIOGLUCOISQ.IERASE . . 56
6. ACTIVITY OF 6-PHIOSPHOFRUCTOKINASE . . 58
7. ACTIVITY OF ALDOLASE . . . . . . 60
8. ACTIVITY OF GL CERALDEITfDE-3-P:IOSP:iATE
DE1TDROGQENASE .............. 63
9. ACTIVITY OF ENOLASE . . . . . . 68
LIST "" FIGURES
1. T2ll CATAMOLISA OF GLUC S . . . . 3
2. TIE. RFDUCTIO:i OF TP:J ; GLUC( -6-P ..ATT
DL. ij; COGrI..S I CILL-FR I XT AC' . 3
3. T .7 RLDUCTI J OF TPA ': 6-P; ;T *'J -'C TL
DEiDT,;7G-SE I>,: CELL-Fill iXT FCT . 53
4. ALDOLASE . . . . . . . .. 61
5. Ti'E REDUCTION CF DP: GL':ClRALDi .lDr-3-
P:.JSPIATL D-I 7DRXOGIASL I;i AC T .J -
EXTRACTS ............... 61
6. GL CERALDI Di -3--i' PiIAT r 64
7. Pi! -i'-:,L C KL .S . . . . . 6
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
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 (Zea mayg L.) by increasing the participa-
tion of the pentose phosphate pathway in glucose catabolism
(Figure 1). Their ij vivo 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 was undertaken to evaluate the effect
or effects of 2,4-D on the in 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 pathway
(Figure 1) or both, which results in a shift of the major
pathway of glucose catabolism. To test this hypothesis,
the activities of the enzymes of the control, i.e. buffer
treated etiolated corn seedlings, were 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 1)
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 1).
starch- ----glucose-6-phosphate- >-fructose-6-phosphate
6-phospho- P 1
gluconolactone erye- fructose-1,6-phospha
L 4-phosphate D
6-phospho- glyceraldehyde- E dihydroxy-
cluconate 3-phosphate acetone
sedoheptulose- ; phosphate
S \ 2 carbon
\ N / pool G
E. triose isomerase
G. phosphoglyceric kinase
H. phosphoglyceric mutase
J. pyruvic kinase
Pentose phosphate pathway enzymes:
Figure 1. THE CATABOLISM OF GLUCOSE.
~ _~ ____ __________ ~I~
_I_ ___________I ~I~
_ __ ____ _ ~_____ ~~m~
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-forties. 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. But 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 growth. While 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
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
After considering the factors cited above, it was
decided to limit this review of literature to research
papers which deal with the probable initial mode or modes
of action of 2,4-D and several physiological responses to
Effects of 2,4-D on Gas Exchange
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, pil 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
(88,89,114,159) 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 qf 2,4-D on Mineral Metabolism
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 (N) 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 (lycoper-
sicon esculntm n Mill) (126,127) were treated with 2-
methyl-4-chlorophenoxyacetic acid ('CPA) and tobacco
(Nicotiana tabacum L.) with 2,4-D (164). Cooke (33)
stated that within the first 24 hours after treating beans
(Phas-eou vulgaris L.) with 2,4-D, the uptake of potassium
was markedly increased, but that after 24 hours this uptake
was markedly inhibited. lie 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
Treatment with 2,4-D has been observed to inhibit the
accumulation of potassium nitrate (KNO3) in excised wheat
roots (Trjitium spp) (114), and to promote the accumulation
of the salt in sugar beet leaves (Be vulgaris 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. (165) 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. 3erg and
AcElroy (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 (Avena sativa L.) the nitrogen uptake is only slightly
decreased, 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 of results
probably is caused by the same factors which were listed
as influencing gas exchange measurements. Increased
phosphorus uptake was observed (160) and increased inorganic
phosphorus content of the entire plant (101), 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 Butts (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 follows 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
incorporation of p32 into glucose-l-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. Ormrod and Williams (118) 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
plant to determine the initial mechanism of action of
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 (P.E) (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). 'ethylation of
pectin is thought to increase wall plasticity by reducing
the number of carboxyl groups which may be cross-linked by
divalent cations (1). Ordin et al. (117) presented data
which indicate that indole acetic acid (IAA) increased the
formation of methylesters of pectin. These data support
the hypothesis that esterification of carboxyl groups of
pectin is involved in the mechanism of cell expansion.
Bryan and Newcomb (21) noted that IAA stimulated the
activity of P.E above the control level. Glaszious and
Inglis (60,61,62) presented data which indicate that auxins
are effective in binding PME tc cell wall preparations.
PAE 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:AE,
it should be insensitive to metabolic conditions such as
temperature and presence of oxygen. The work of Adamson and
Adamson (1) 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 (11) noted that the chelating
substance ethylenediaminetetraacetic acid (EDTA) would
act as a growth substance by stimulating the extension
growth of Avena coleoptiles. Working 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 (,Ag) 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 O. D. were not due entirely
to chelation. The general status of this theory today is
that chelation could affect certain enzymatic reactions
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
Effects of 2,4-D on the Metabolism of Carbon Compounds
and Related Enzyme Systems
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 Avena coleoptile sections, merger
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 IAA present. Thus, they postulated a
role of enzyme activator for auxins. Many research
workers presently are continuing work on this basic idea,
although they do not agree as to what area of metabolism
is affected. Two 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
influence changes in cell structure and subsequent water
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 (Pslum 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
proteins may be important in explaining auxin action.
Gordon (63) reported some results which indicated IAA
may be associated with proteins as an absorbed, unstably
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 C1402 and found a large increase
in the incorporation of C14 into aspartic and glutamic
acids. They also noted a decrease in photosynthesis.
Thus, they postulated more CO2 enters through the Krebs
cycle with a subsequent increase in amino acids. Boroughs
and Bonner (17) found that IAA did not affect the incor-
poration of labeled glycine or leucine into proteins of
corn and Avena 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
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
that nucleic acids are key components in the control of
growth. Skoog (139) presented a good discussion of the role
of nucleic acids in growth and presented a hypothesis link-
ing IAA action with nucleic acid metabolism. He gave
experimental results in which both deoxyribonucleic acid
(DNA) and ribonucleic acid (RNA) are increased in tobacco
pith tissue treated with IAA (136). Croker (35) studied
the effect of 2,4-D on mitosis in Alliun 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
growth-regulators and ionizing radiation. As further
evidence of their participation in growth, Rasch et al.
(122) noted that both RNA and D:JA 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
RNA 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 P32 or C14 with and without IAA
in a study of the incorporation of labeled compounds. After
two hours the nucleic acid fraction of the sections was
isolated and the radioactivity determined. The activity
was taken as an indication of the effect of IAA on in-
corporation and thus the effect of IAA on the cellular
metabolism. D*A and RIJA fractions from IAA treated tissue
incubated with p32 counted higher, indicating that IAA
affected the synthesis of nucleotides composing the
nucleic acids. When incubated with labeled acetate,
format or glycine, IAA treatment did not affect the
counts. Hle concluded that IAA stimulates the synthesis
of nucleotides and phosphorylation reactions as evidenced
by the tracer studies; while the compounds labeled with
C 4, 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 or 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. When 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). 'Aarre and Forti (104)
states that the primary effect of auxin does not depend
upon phosphate acceptor availability, but more probably
involves the activation of oxidative enzymes. French
and Deevers (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
RNA. HIe concluded that growth induced by auxins involves a
growth 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) was 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 (Ricinus communis Linn.)
was inhibited as much as 70 percent by 2,4-D (66). Kvamme
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. Wort 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 vitro by 2,4-D (18,151). Neely et al. (115)
reported that the activity of both alpha and beta amylase
was inhibited by 2,4-D. Salivary amylase was also reported
to be inhibited in vitro by 2,4-D (156), but crystalline
human amylase was reported to be non-responsive to 2,4-D in
vitro (45). Most of the work with polysaccharides indicates
that 2,4-D treatment increases their utilization in vivo.
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
The changes in sugar content of 2,4-D treated plant
tissues have been reported by numerous workers. '4itchell
and Brown (111) reported that sugars in 2,4-D treated
morning-glory (Ipomoea spp) plants increased above the
controls at first, but then decreased and were nearly
depleted by the third week following treatment. Using
dandelions (Taraxacum officinale w.), Rasmussen (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. lie 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
(Faqopyum esculentum A.) sprayed with 2,4-D were analyzed
for total sugars by Wort (167). Ile 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
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 were 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
RPbinson (140) incubated tobacco stem segments with various concentrations
of IAA for several months. They noted that reducing sugar
content increased in all cases.
In 1956, Humphreys and Dugger (78) began a series of
experiments on the effects of 2,4-D on plant metabolism.
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 was 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 1402 evolved when glucose-l-C14 and
glucose-6-C14 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 1402
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
pathway, the rate of C1402 production from the first
carbon of the glucose molecule would initially be greater
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 '
causing more lucose 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 DAP promoted catabolism 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
103 nolar (.4) 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 v riation in the results of rang et 1.
and those of ;uAphreys and : er could be caused by
several factors: :u ipLreys and '-er's results .iere
obtained ixnediately follow' 2,- treatnet, ereas
:anj et al. used tissue .'hich had been treated seven
day prior to the study; difference in plant tissues
used; and the point made by Humphreys and Dugger (82),
that evaluation of catabolic pathway of glucose based only
on the yield of C402 from labeled glucose is not possible.
Thus, the work 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
Plant materials. 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 220 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, 10-2 M.
The other group of seedlings was treated by immersing
the roots in buffer plus 2,4-D, 10-3 :. The trays were
placed in the dark for 12 hours at 220 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 extracts were prepared from
each group. The same procedure was followed for both
2,4-D and buffer treated roots.
Ten grams of excised roots were added to 100
milliters (ml) of water (4-60 C) and placed under
refrigeration for about 30 minutes. The water was
decanted off and the roots were placed in an ice-cold
mortar containing 10.0 ml of 0.05 : tris hydroxymethyll)
aminomethane (Tris), pH 7.4 plus 1.0 ml of 1.0 A. 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 00 C.
The yellowish-brown supernatant fraction was de-
canted and its pH adjusted to 7.0 with dilute sodium
hydroxide (:;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 M
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 powder extracts is essentially that given by
Hageman and Arnon (65). Ten grams of corn roots were
added to 100 ml of water (4-60 C) and placed in the
refrigerator for about one hour. The water was decanted
off and the roots were ground until no intact roots
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 EDTA. Cold acetone (125 ml, -150 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 (-150 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
(P205) 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 Whatman number
one filter paper. The resulting filtrate was designated
as the acetone powder extract. All extracts were used
the same day they were prepared.
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.
Studies on the Pentose Phosphate Pathway
Pentose disappearance. 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 380C. The reaction was started by adding the cell-free
or acetone powder extract. Each reaction mixture consisted
of: 0.2ml of triphosphopyridine nucleotide (TPN:) (4 milli-
grams (mg)/ml); 0.1 ml of flavinadenine mon-nucleotide
(F.UI) (1 mg/ml); 0.1 ml of .gC12 (0.1 A); 3.4 ml of Tris
buffer (p1H 7.4, 0.1 ..); 0.2 ml of R-5-P (0.1 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 (1.0 or 2.0 ml) of the
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 .Aejbaum (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-
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
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. HIorecker and Smyroniotis (74) in-
dicated that fructose interfered with the pentose deter-
mination, but in these preliminary experiments it did
Chromatographic studies on 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 380C for 30 minutes with 1.0
ml of alkaline phosphatase (1 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
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. When two-
dimensional chromatographs were run, water-saturated
phenol was the second solvent. The orcinol spray of
Klevstrand and Nordal (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 unknown sugars.
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.
Glucose-6-phosphate dehydrogenase. 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 + TPNW--6-PHOSPHOGLUCONATE + TPNH- H.
The assay mixture contained the following components:
2.0 ml of Tris buffer (pH 7.4, 0.1 .A); 0.1 ml of TPN
(4 mg/ml); 0.1 ml of .AgCl2 (0.1 .1); 0.1 ml of G-6-P
(0.1 A); cell-free extract; and water to 3.0 ml total
volume in Beckman corex cells of one centimeter (cm)
light path. TPN 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.
6-Phosphoqluconate dehydrogenase. The presence of
this enzyme was studied by measuring the reduction of TPNI
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 .4) was used as the substrate in the reaction given
6-PGA 4- TPi-IP-RIBULOSE-5-PHOSPHATE + CO2+TPNH+H.
Stdies on the Glycolytic Pathway
PhosphoQlucoiomerase. 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:
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 .);
0.2 ml of G-6-P (0.1 .I); and 0.1 ml of cell-free extract.
The mixtures were incubated for ten minutes at 380C. The
reaction was stopped by adding 3.5 ml of 8.3 A. IC1. One
ml of 0.1 percent resorcinol in 95 percent ethanol was
added and the mixture heated for ten minutes at 800 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
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
6-Pphshofructokinase 6-phosphofructokinase, which
catalyzed reaction 1 below, was measured by the procedure
of Ling et al. (98), using the complete system shown in
equations 1, 2 and 3 below. In this system, F-6-P and ATP
are substrates, and 'Ag is an essential co-factor.
1. F-6-P ATP----F-1,6-P + ADP.
2. F-l 6-P-4 DIHYDROXYACFTO'E PHOSP IATE -
GL iC RALDEHYDE-3-P1 OSPHATF.
3. DIhYDROXYACETONE PHOSP!ATE -1 DP:JHI--ALPA4-
GLCL.ROPHOSPHATE -+ DP:J.
The fructose-l,6-phosphate (F-1,6-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;JI) to alpha-glycerophosphate.
The oxidation of DP'!H in reaction 3 was followed with a ?eck-
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 (pil 8.0, 0.2 '1); 0.3 ml of ATP (0.1 .);
0.2 ml of F-6-P (0.1 .) ; 0.3 ml of AgCl2 (0.01 .i); 0.1 ml of
cysteine-hydrochloride (0.2 A) ; 0.1 ml of DP;1H (2 mg/ml); 0.lml
of aldolase (1 mg/ml); 0.05 ml of alpha-glycerophosphate
dehydrogenase (1 mg/ml); 0.1 nl of cell-free extract; and
water to 3.0 ml total volume. The blank consisted of the
complete reaction mixture as given above, minus DPNH. 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 Beck (10),
in which the triose phosphates formed in the reaction below
are trapped with hydrazine:
F-I 6-P <---DIHYDRCKYACETONE PHOSPHATE+
The triose phosphate hydrazones formed were treated
with alkali, and the color produced by the addition of
2,4-dinitrophenylhydrazine (2,4-DIIPH) and NaOH was read
with a Klett colorimeter using filter number 54. A
typical reaction mixture consisted of: 1.0 ml of Tris
buffer (pHl 8.6, 0.1 .); 0.25 ml of F-I, 6-P (0.05 :i);
0.25 ml of hydrazine (0.22:.); 0.2 ml cell-free extract;
and 0.8 ml of water. The reaction mixtures were incu-
bated at 380C for ten minutes with various amounts of
extract. Results of the assay are presented in a graph
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-3-phosphate dehydrogenase. Activity
of glyceraldehyde-3-phosphate dehydrogenase (GPD1i) could
not be demonstrated in cell-free extracts; therefore,
acetone powders prepared following the basic method of
Hageman and Arnon (65) were used to study the activity
of this enzyme. GPDII was measured in a Beckman DU
spectrophotometer by the optical test of Warburg and
Christian (158) based on the increased O.D. of reduced
DPN and TPN at 340 mu in the reaction below:
GLYCERALDEHYDE-3-PHOSPHATE 4 DPNI' + J PO04< -
1,3-DIPHOSP IOGLYCERATE -t DPNH + H .
The following reaction mixtures were prepared in Beckman
corex cells of one cm light path: 0.2 ml of DPN (4 mg/ml);
0.1 ml of sodium arsenate (0.17 ;I); 0.1 ml of potassium
flouride (0.1 'M); 0.15 ml of reduced glutathione (0.1 .'4);
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 .A); 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
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
N versus time.
Phosphoglyceric kinase. 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 with 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 MgCl2 (0.015 ;); 0.5 ml of
3-PGA (0.052 M); 0.2 ml of ATP (0.1 :;); 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 (FeCl3) -HCL-TCA mixture was
added prior to adding the extract. The reaction mixtures
were incubated at 370 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 FeC3-I-Cl-TCA mixture.
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
1. CH3* CO COOH- 3-CH3' CHO + CO2
2. C3 CO COOHI + CH3* CHO -< CH3- CO CHOH CH3+
3. 2 CI3* CHO-<->CH3* CO CHOII C13.
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
CO2 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 l,l-dimethyl-3,5-diketo-
cyclohexane (0.05 'A); 0.1 ml of thiamine pyrophosphate
(ThPP) (5.8 X 10-4 .l); 0.2 ml of ;IgCl2 (0.01 .0); 0.1 ml
serum albumin (one percent); cell-free or acetone powder
extract; water to 2.8 ml total volume; and in the side arm,
0.2 ml of sodium pyruvate (0.5 4). At zero time, the
pyruvate was tipped into the main compartment and CO2
evolution was followed for periods up to two hours.
Phosphoglyceric mutase, enolase and pyruvic kinase.
This group of enzymes catalyze reactions 1, 2 and 3,
1. 3-PHOSPHOGLYCERATE ---~2-PHOSPHOGLYCERATE.
2. 2-PHOSPHOGLYCERATE--- PHOSPHO-ENOL-PYRUVATE+ H20.
3. PHOSPHO-ENOL-PYRUVATE + 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 Boyer (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 I4); 1.5 ml of Tris buffer
(pH1 7.4, 0.1 M4); 0.2 ml of MgCl2 (0.1 .4); 0.2 ml of ADP
(0.1 .); 0.1 ml of 2,3-diphosphoglycerate (2,3-DPGA)
(30 mg/5 ml); 0.1 ml of KC1 (0.5 .A); 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
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)
to pyruvate: 1.5 ml of Tris buffer (pH 7.4, 0.1 A);
0.2 ml of 2-phosphoglycerate (2-PGA) (0.1 .A); 0.2 ml
of .AgCl2 (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 380 C.
Pyruvate was determined after the reactions were stopped
with 2,4-DNPH. The results of colorimetric determinations
of pyruvate are presented on the basis of Klett readings
per mg N 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 ..); 0.2 ml of PEP
(0.05 A4); 0.2 ml of MgC12 (0.1 M); 0.2 ml of ADP (0.1 '4);
0.1 ml of KC1 (0.5 :4); 0.1 ml of extract; and water to
2.5 ml total volume. The reaction mixtures were incubated
for ten minutes at 380 C. Reactions were started by adding
enzyme extract and stopped by adding 2,4-DNPH. The blank
consisted of a complete reaction mixture minus PEP, which
was added after the 2,4-DNPH. The reaction mixtures then
were assayed for pyruvate. .AcCollum 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 + 1120 <--'PYRUVATE + I:IORGANIC PHOSPHATE.
Phosphatase activity was 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
Reagents used 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-1,6-P from lMann Research Laboratories,
Incorporated; F-6-P and sodium pyruvate from Nutritional
Biochemicals Corporation; DPN and DPMIH 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 TPN, TPJIH,
6-PGA, F-6-P, ADP, PEP, 2-PGA and F:-N from Sigma Chemical
Company, Sedoheptulosan was generously donated by Dr.
No 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 'Aann Research Laboratories, Incorporated; alkaline
phosphatase from Nutritional Biochemicals Corporation; and
alphaglycerophosphate dehydrogenase from Sigma Chemical
Company. Bovine serum albumin was obtained from .'ann
Research Laboratories, Incorporated.
The 2,4-D was neutralized with NaOH 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
excess of potassium sulfate to a solution of the barium
salts in dilute acid: G-6-P, R-5-P, F-6-P, F-1,6-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 ZC1 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.
Studies on the Pentose Phosphate Pathway
Pentose disapEearance. The enzymes which 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 N per six ml. The percent change due to
2,4-D is based on the number of umoles per mg N per six ml.
-t V4 o0. anp
GCI-' 01. anp
C-tl4Z o0 anp
X-4 CN ) n 4 Lo
0 I0 0 I0 0I
I I I I
0 0 ICM CM co
in .. 1.
w) l-4 lIC 1V) I
3 3 1 ID I
II I I- ,-
I U) Qu I Q. a i
-n CM--4 \ O- m CN
Id iO Iq IO IU
II I I 0 I 0
- ) IO I n In I n
a L a -I I
I O) 1O 1O IO MO I
* in * .* I
ID Io (
IU) I U
00 -n I
0 4 0
*t IO *
II 10 I
I I I 4J I
II I 1
* -0 *0
0 a 0
.3 C -
*o k 4
COO) > 0
x ) < -0-
o ( c )
O O0 c'
Ur 4-o 4-
0 > 4 0
4- U CO -
%-POC > 4
:3 m 0P -4
04-' > *-H
CH C C10
*a CM 0
F- c0 o
0 (f o o
O C C
if) OO n 0
*H1 r a n 1
QZkf-1 4- ^
The reaction mixtures contained 20 umoles of pentose per
six ml at time zero. The pentose utilized (Table 1) in
the 60-minute incubation period varied from 2.4 umoles
(Experiment 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 1) 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 1), with the
increase ranging from 8 to 32 percent in Experiments
1 through 5. The formation of heptulose also was 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.
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
TPN on the reaction was studied. No effect was noted,
with or without TPN, 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
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
0 0 0
S V 0
- 4 C') T n O \0 r-
om n-: r cc O co cr O
. . . " " d*
=NQ M NO ) O 0
00 00 00 00 00 00 00
ON Q'D C o7 w w n On r-r-- m 'T r- O\
Om o on \o \0O 0 Ow \0 (0
*I* * * * . *
C" O1 4 r- _- -4
q\o O w0 N 0 w It O C q N0
P-4o o o On n oo qd d Lo
NH- -IN nC C.-Y OM4 m \ 0m On NmO
Cn On 6 O CM OOC O6 6 cMO
N m mm N C HY y N CN 01)
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.
Chromatographic studies on pentose disappearance.
These studies were designed to support the studies on the
disappearance of R-5-P. Chromatographic studies following
the 60-minute 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 B-naphtholamine. 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.
Glucose-6-phosphate 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 Deckman DU spectrophotometer at 340 mu.
Spectrophotometric assay for this enzyme was com-
plicated by the formation of 6-PGA which was the substrate
for 6-phosphogluconate dehydrogenase which also reduced
TPN, 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
ACTIVITY OF GLUCOSE-6-PHOSPHATE DEIHDROGENASE*
Experiment Treatment** Change in O.D. per mg % Increase
Number 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: TPJ, 400 ug; MgCl2,
10 uM; G-6-P, 10 uMA; Tris buffer, pH 7.4, 200 u.; 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.
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
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. Buffer
treatment resulted in an average change of 0.75 in O.D.
per mg protein per five minutes, while the 2,4-D treat-
ment resulted in an average change of 0.94, thus indicating
that in vitro G-6-P is oxidized faster following 2,4-D
6-Phosphogquconate dehydrogenase. 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
I I I L
o 0 tU 0 o U 0
') r CJ \J -
N Bwu/(nwuo~t)AJISN30 VDIldO
_I I_ I I
S u) 0 91 0
N 6uw/(nwuot,)AJISN30 7'VOlJdO
) 0J U) w-4
0 U C.
U -- w)
4 U) L *
\nL.0 w C
L > .
U-l W u- *
C3 I *
ACTIVITY OF 6-PHOSPHOGLUCOIATE DEHfDROGENASE*
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; MgCl2,
10 u.'l; 6-PGA, 10 uM; Tris buffer pH 7.4, k00 uA; 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.
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-6-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
These in vitro 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
Studies of the Glycolytic Pathway
Phosphoglucoisomerase. 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 are easily within the range of experimental error.
ACTIVITY OF PHOSPHOGLUCOISO.1ERASE*
Experiment Treatment** ui_ of F-6-P produced per ma N
Number 10 min. 1 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 u.',; 0.05 ml of cell-free extract;
and to 0.5 ml total volume with water. These were incubated at
38 C for the prescribed periods of time.
**See Table 1.
Therefore, from the results of these in vitro studies with
cell-free extracts, 2,4-D treatment does not affect the
activity of phosphoglucoisomerase.
6-Phqsphofructokinase. Quantitative measurements of
the activity of 6-phosphofructokinase were obtained by
incubation of F-6-P, ATP and M:gCl2 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
N 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
Aldolase. Aldolase activity in cell-free extracts
was measured by trapping the triose phosphates formed
when F-1,6-P is cleaved by aldolase. In extracts from both
TAi LE 6
ACTIVITY OF 6-PHOSPHOFRUCTOKIIHASE*
Experiment Treatment** Change in O.D. per 7 Decrease
'urnber mg per min.*** Due to 2,4-D
-Complete reaction mixture contained: Tris buffer, pH 8. ,
100 u. ; ATP, 30 u.; F-6-P, 20 u.i; ..gC12, 3 u.; cysteine-hydro-
chlorid, 20 u..; DP;:iH, 220 ug; aldolase, 10 ug; alpha-glycero-
phosphate dehydrogenase, 50 ug; and water to 3.0 ml in corex
ieckman cells with 1 cm light path. O.D. readings at 340 mu.
**See Table 1.
***-Corrected for endogenous.
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.
Glyceraldehyde-3-phosphate dehydroqenase. Attempts
to demonstrate GPDH 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 DPN-dependent enzyme. The lack of a TPN-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 :Marcus (103), that TPN triose phosphate dehydro-
genase is controlled by the photomorphogenic reaction.
Neither DPNH nor TPNH was oxidized in incubation
mixtures containing all of the constituents used to study
the reduction of DPN.
Neither cysteine nor glutathione was 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
ACTIVITY OF ALDOLASE*
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 380C for
10 min., stopped with 0.75 ;i NaOl, and the triose phoshpate
hydrazones measured: Tris buffer, plH 8.6, 100 u.; F-1, 6-P,
12.5 u.M; hydrazine, 55 u.1; 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.
N O O
< 0 -I
O H (
_- -j 0 x
E -H 0 C
C ~ c
a O. l
r U *4
*C -P c *H
"- 0 +
o o Cr o
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
ACTIVITY OF GLYCERALDEHDEDE-3-PHOSPHATE DEHYDROGENASE*
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 uM:; DPN, 800 ug; sodium arsenate, 170 u'A; potassium flouride,
10 uM'; reduced glutathione, 15 uM; glyceraldehyde-3-phosphate,
2000 ug; water to 3.0 ml total volume; and acetone powder
extract. Blanks omitted DPJN. Enzyme extracts were added to start
the reaction and the increase in O.D. at 340 mu was followed at
**See Table 1.
0 0 0
O O O0
N bwlo/9NiGV3 i.
(o' )t I 1 dO
0 = *-3 4J
.o -- -
.C: r-4 X :
u U) U U
0 (D F -4
.o co H <
-- H H -- 0 -
4 I C o
.CO 0 0
0. 0 40C 04
3~o +-, ("l >
S0 3CO (
00 -0 *
i- a- .-
o -- L *4- Q
0 0r0 *0
E a) 0
0 .4 (D C C:
\~ ~ Q nu oc
\ o ^i *.lQa OO
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 GPD!I bound DPI. GPDH 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
DP:I per mole of enzyme. Apparently both DP:;J and DPN are
bound by the enzyme and both have different dissociation
constants. Stockwell (146) states that DPNH has about
one tenth of the affinity of DP:J for binding sites on the
enzyme. Other work suggests that DP.I is strongly
bound. Thus the slower linear rate after about two minutes
of reaction time is probably controlled by the rate of
dissociation of DPIHI. These slower linear rates are not
additive with increased aliquots of extracts. This may be
explained on the basis of a build-up of DP: N, which competes
with DPN 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.
Phosphoglyceric kinase. The activity of phospho-
glyceric kinase was 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
in these experiments. Obviously, 2,4-D treatment of corn
seedlings did not affect the in vitro activity of phospho-
Carboxylases. No 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 two hour incubation period. This infor-
mation allowed an assay for the activity of phosphoglyceric
mutase, enolase and pyruvic kinase by measuring pyruvate.
Phosphpolyceric mutase. 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.
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 was 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.
Pyruvic kinase. 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 liability of the enzyme was noted in these experi-
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
ACTIVITY OF ENOLASE*
Experiment Treatment** Klett readings
Number per mg protein
26 Buffer 20
27 Buffer 36
28 Buffer 27
*The reaction mixture contained: Tris
buffer, pH 7.4, 150 u.1; 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.
except in a few cases. When 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 25 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 liability and the high endogenous phos-
These in vitro 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
In a study of the in vitro 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. When si-milar
extraction procedures are followed with both treated
and untreated tissues, are similar percentages of each
enzyme extracted from both tissues? Second, in Ji vitro
work, changes could occur in an enzyme or enzymes as a
result of the extraction procedure. .'ote 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
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, pH 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 'I or per mg protein, this
could skew the results. In 50 consecutive :N determinations,
2,4-D treated tissues had an increased 1N content in 20
determinations. In 16 determinations, buffer treated
tissues had the highest "4, 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 N determinations
affect the trend of the observed results of an experiment.
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 when 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. When 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
Forsman (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 growth-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
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 was noted
on both dialyzed cell-free extracts and undialyzed acetone
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 vivo 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 D vitro study of the enzyme activity of
both the pentose phosphate pathway and the glycolytic
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 were 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 pathway be favored over the glycolytic pathway?
It is difficult to see why such small changes can affect
the metabolism of a plant. Note should be taken of the
fact that glycolytic enzymes were 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 Hess (24,25,26,27,
28,29,72) also have studied metabolic control mechanisms
using ascite tumor cells and bakers' yeast cells.
Wu 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
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-
In this series of experiments, the enzymes of both
glycolysis and the pentose phosphate pathway were present
in the extracts. Even though previous in vivo 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.
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
TPiN or of electron acceptors for reduced TP1. So any
action whereby TPN is formed, either by synthesis or by
oxidation of reduced TPN, may stimulate the pentose
phosphate pathway. Action of growth regulators through
nucleic acid metabolism was proposed and somewhat
substantiated by Skoog (136,139,140). Key et al. (92),
working with 2,4-D treated soybeans, concluded that 2,4-D
may affect the metabolism of nucleotides which are involved
in mitochondria growth. 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-
bolisn. 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
which were oxidizing glucose via the pentose phosphate path-
way 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 pyrinidine 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.
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 IIess (30), whereby small differences can be
measured accurately in short-time intervals employing
SU.AAARY AND CONCLUSIONS
A series of experiments was conducted to study the
effects of 2,4-D on the n vitro 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
Three-day-old etiolated corn seedlings were divided
into two groups and treated either with 10-2 A phosphate
buffer (pH 5.3), or with buffer plus 10-3 M 2,4-D for
12 hours at 220C. After the 12-hour treatment period,
the seedlings were 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 powder
extracts as the enzyme source. Individual enzymes were
studied in reaction mixtures containing excess substrate
plus the known cofactors, with enzyme concentration being
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
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 jD vitrg studies support the in vivo
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
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.
I :.LIOGRAP .
1. Adamson, D. and :i. 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
metabolism. VI. Effect of 2 4-D on the meta-
bolism of aspartic acid and glutanic acid in the
bean plant. Plant Physiol. 31:34-37. 1956.
3. Armarego, W. L. F., !. J. Canny and S. F. Cox.
4etal-chelating properties of plant- growth sub-
stances. Nature 1 1:1176-1177. 1959.
4. Asana, R. 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. C steine-H2SO4 reaction for hexose.
In methods in Inzymology III. S. P. Colovick
and ". i'. iCaplan, d. pp. 81-_ ew .ork:
Academic Press. 15/7.
6. Avery G. S., Jr. Stimulation of respiration
in relation to growth. In Plant Growth Sub-
stances. F. Skoog, Id. pp. 105-109. ladison,
Wisconsin: University of Jisconsin Press. l. l.
7. Axelrod, and R. S. andurski. Phosphoglyceryl
kinase in higher plants. J. iol. Chem. .04:
9 -4 1 ~13.
8. .'il, 1. Die Diagnose der Pentosurie. Deut.
med. .Voch. 28: 3-2 4. 1 -02.
9. .ass, S. T. C. L. : mner and !. Sell. Effects
of 2,4-dichiorophenoxy cetic acid on the mineral
contents of cranberry bean plants (Phaseolus
vul aris) -ich. State Univ. A xp. Sta. rt.
.-ull. 42:43-46. 1 9.
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.
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.
Wightman, Ed. pp. 284-291. New York: Academic
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 Avena 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. B. and S. P. Sen. Relationship between
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, H. and J. Bonner. Effects of indoleacetic
acid on metabolic pathways. Arch. Biochem. Biophys.
18. Erakke, A.. K. and L. G. Nichell. Lack of effect
of plant growth-regulators on the action of alpha-
amylase secreted by virus tumor tissue. Bot. Gaz.
19. Brody, T. M. Effect of certain plant growth sub-
stances on oxidative phosphorylation in rat liver
mitochondria. Proc. Soc. Exptl. Biol. M.ed. 80:
20. Brown, J. W. Effect of 2,4-D on the I-oO relations,
the accumulation and distribution of slid matter,
and the respiration of bean plants. Bot. Gaz.
21. Bryan, W. HI. and E. H. Newcomb. Stimulation of
pectin methylesterase activity of cultured tobacco
pith by indoleacetic acid. Physiol. Plant. 7:290.
22. Burstrom, 11. 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 E. K. Ng. 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 Baker's yeast.
J. Biol. Chem. 234:3036-3040. 1959.
25. Chance, B. Phosphorylation efficiency of the intact
cell. III. Phosphorylation and dephosphorylation
in yeast cells. J. Biol. Chem. 234:3041-3043. 1959.
26. Chance, B. and B. Hess. Metabolic control mechanisms.
I. Electron transfer in the mammalian cell. J. Diol.
Chem. 234:2404-2412. 1959.
27. Chance, B. and B. Hess. Metabolic control mechanisms.
II. Crossover phenomena in mitochondria of ascites
tumor cells. J. 3iol. Chem. 234:2413-2415. 1959.
28. Chance, 3. and B. Hess. Metabolic control mechanisms.
III. Kinetics of oxygen utilization in ascites tumor
cells. J. Liol. Chem. 234:2416-2420. 1959.
29. Chance, B. and B. Hess. Metabolic 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, B. 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. B. Ginsburg and C. Heitner-Wirguin.
Metal-chelating 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.
36. Datta, S. C. and S. Dunn. Effects of light qual-
ity on herbicide toxicity to plants. Weeds 7:55-65.
37. Dische, Z., L. B. Shettles and :'. Osnos. New specif-
ic color reactions of hexoses and spectrophotometric
micromethods for their determination. Arch. Biochem.
38. Eliasson, L. Growth response of pea roots to 2,4-D
applied to the hypocotyl. Physiol. Plant. 12:834-
39. Eliasson, L. Inhibition of the growth of wheat
roots in nutrient solutions by substances exuded
from the roots. Ann. Royal Agr. Coll. Sweden.
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 B. L. Brannaman.
Influence of pIH on 2,4-dichlorophenoxyacetic acid
and acetic acid activity in Chlorella. Plant Physiol.
42. Fang, S. C., F. Tenny and J. S. Butts. Influence of
2,4-dichlorophenoxyacetic acid on pathways of glu-
cose utilization in bean stem tissues. Plant Physiol.
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.
44. Fawcett, 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 B. ii. 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.
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. Bot. Gaz.
51. French, R. C. and H. Beevers. Respiratory and
growth responses induced by growth regulators and
allied compounds. Amer. J. Bot. 40:660-666. 1953.
52. Friedemann, T. E. and G. F. Ilaugen. 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. Bot. Gaz. 118:130-133. 1956.
55. Galston, A. VI. 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.
56. Galston, A. 'W. and W. K. Purves. The mechanism of
action of auxin. Ann. Rev. Plant Physiol. 11:239-
57. Gatt, S. and E. Racker. Regulatory mechanisms in
carbohydrate metabolism. I. Crabtree effect in
reconstructed systems. J. Biol. Chem. 234:1015-
58. Gatt, S. and E. Racker. Regulatory mechanisms in
carbohydrate metabolism. 1I. Pasteur effect in
reconstructed systems. J. Biol. Chem. 234:1024-
59. Gibbs, A. Triosephosphate dehydrogenase and glucose-
6-phosphate dehydrogenase in the pea plant. Nature
60. Glaszious, K. T. The effect of auxins on the binding
of pectin methylesterase to cell wall preparations.
Aust. J. Biol. 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. Mokrasch and V. D. Hospelhorn.
Adenosine mono-, di- and triphosphate, pyruvic kinase,
hexokinase and polynucleotide phosphorylase assay.
Biochem. 3iophys. 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.
66. Hagen, C. E., C. O. Clagett and E. A. Helgeson.
2,4-dichlorophenoxyacetic acid inhibition of castor
bean lipase. Science 110:116-117. 1949.
67. Hanson, J. :. and J. Bonner. The relationship between
salt and water uptake in Jersulem artichoke tuber
tissue. Amer. J. Bot. 41:702-710. 1954.
68. Heath, O.V S. and J. E. Clark. Chelating agents
as plant growth substances. A possible clue to
the mode of action on auxin. Nature 177:1118-1121.
69. Heath, O. V. S. and J. E. Clark. Chelating agents
as growth substances. Nature 178:600-601. 1956.
70. Heath O. V. S. and J. E. Clark. Comment on the
article by Armarego. Nature 183:1177. 1959.
71. Henderson, J. H. .., 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. Science120:710-712. 1954.
72. Hess, B. and B. Chance. Phosphorylation efficiency
of the intact cell. I. Glucose-oxygen titrations
in ascites tumor cells. J. Biol. Chem. 234:3031-
73. Hiatt, H. H. and J. Lareau. Studies of ribose meta-
bolism. VIII. Pathways of ribose biosynthesis in
vivo and in vitro in rat, mouse, and human tissues.
J. Biol. ieem. 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. Nature 178:255-256. 1956.
76. Howell, R. W. The inhibiting effect of root tips
on the elongation of excised Pisum epicotys cultures
in vitrl. 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.
78. Humphreys, T. E. and W. 'A. Dugger, Jr. The effect
of 2,4-dichlorophenoxyacetic acid on the respiration
of etiolated pea seedlings. Plant Physiol. 31
79. Humphreys, T. E. and W. A. Dugger, Jr. The effect
of 2,4-dichlorophenoxyacetic acid on pathways of
glucose catabolism in higher plants. Plant Physiol.
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-
81. Humphreys, T. E. and W. A. 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 W. 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.
83. Johnson, E. J. and A. R. Colmer. Relationship
between magnesium and the physiological effects of
2,4-dichlorophenoxyacetic acid on Azotobacter
vinelandii and Rhizobium meliloti. Jour. Bact.
84. Johnson, E. J. and A. R. Colmer. Further studies
on the mode of action of 2,4-dichlorophenoxyacetic
acid on Azotobacter vinelandii as related to mag-
nesium and phosphate. Jour. Bact. 73:666-669. 1957.
85. Johnson, E. J. and A. R. Colmer. The relation of
magnesium ion to the inhibition of the respiration
of Azotobacter viineandii 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
Azotobacter vinelandii. Plant Physiol. 33:99-101.
87. Kachmar, J. F. and P. D. Boyer. Kinetic analysis
of enzyme reactions. I. The potassium activation and
calcium inhibition of pyruvic phosphoferase. J.
Biol. 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. Bot. 36:421-426.
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 occurring growth inhibitor in soybeans
following treatment with 2,4-D. Plant Physiol.
92. Key, J. L., J. 3. 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.
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 allyl sulfide in wild
garlic. Bot. Gaz. 113:119-134. 1951.
95. Kravitz, E. A. and A. J. Guarino. On the effect of
inorganic phosphate on hexose phosphate metabolism.
Science 128:1139-1140. 1958.
96. Krimsky, I. and E. Racker. Glutathione, a prosthetic
group of glyceraldehyde-3-phosphate dehydrogenase.
J. Biol. Chem. 198:721-729. 1952.
97. Kvamme, O. J., C. O. 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. Biochem. 24:321-328. 1949.
98. Ling, Kuo-Huang, W. L. Byrne and H. Lardy. Phospho-
hexokinase. In Methods in Enzymology. I. S. P.
Colowick and N. 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. Auzik. 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., 'A. P. ;orris, J. Garcia and C.
Pagan. 2 4-D affects phosphorus metabolism. Science
102. Luecke, R. W., C. L. Hammer and H. M. 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.
104. :.arre, 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. .cCollum, 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. McCollum, R. E., R. iH. Hageman and F. H. Tyner.
Occurance of pyruvic kinase and phosphoenolpyru-
vate phosphatases in seeds of higher plants.
Soil Science 89:49-52. 1960.
107. McElroy, W. C. and B. Glass, Ed. Symposium on
phosphorus metabolism. I. 3altimore: Johns
Hopkins Press. 1951.
108. :AcElroy, '. C. and 3. Glass, Ed. Symposium on
phosphorus metabolism. II. Baltimore: Johns
Hopkins Press. 1952.
109. .4ejbaum, W. Uber die Bestimmung Kleiner Pento-
semengen insbesondere in Derivaten der Adenyl-
saure. 1. Physiol. Chem. 258:117-120. 1939.
110. millerr I. H. and R. 1H. 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. W. 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. IH. 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. iMoewus, Franz. The action of 2,4-D on deaminating
enzymes. 8th International 3ot. Congress. pp. 149-
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 11. 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:
116. Nickell, L. G. Effect of certain plant hormones
and colchicine on the growth and respiration of
virus tumor tissue from Rumex acetosa. 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 Trifolium 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-
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. A. Klein. Nucleopro-
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. 1947.
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. A.. Sell. The
influence of 2,4-dichlorophenoxyacetic acid and the
phosphorus metabolism of cranberry bean plants
(Phaseolus vulgaris). Plant Physiol. 29:490-491.
126. Rhodes, A. The influence of the plant growth-
regulator, 2-methyl-4-chlorophenoxyacetic acid,
on the metabolism of carbohydrate, nitrogen and
minerals in Slanum lycopersicum. J. Exp. Bot.