Group Title: Some effects of 2,4-dichlorophenoxyacetic acid on the carbohydrate methobolism of etiolated corn seedlings /
Title: Some effects of 2,4-dichlorophenoxyacetic acid on the carbohydrate methobolism of etiolated corn seedlings
CITATION PDF VIEWER THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00097991/00001
 Material Information
Title: Some effects of 2,4-dichlorophenoxyacetic acid on the carbohydrate methobolism of etiolated corn seedlings
Physical Description: vii, 98 leaves ill. : ; 28 cm.
Language: English
Creator: Black, Clanton Candler, 1931-
Publication Date: 1960
Copyright Date: 1960
 Subjects
Subject: Seedlings   ( lcsh )
Dichlorophenoxyacetic acid   ( lcsh )
Plants -- Metabolism   ( lcsh )
Agronomy thesis Ph. D
Dissertations, Academic -- Agronomy -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1960.
Bibliography: Includes bibliographical references (leaves 82-97).
Additional Physical Form: Also available on World Wide Web
Statement of Responsibility: by Clanton Candler Black.
General Note: Typescript.
General Note: Vita.
 Record Information
Bibliographic ID: UF00097991
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000469606
oclc - 36795207
notis - ACN4333

Downloads

This item has the following downloads:

PDF ( 3 MBs ) ( PDF )


Full Text









SOME EFFECTS OF

2,4-DICHLOROPHENOXYACETIC ACID ON

THE CARBOHYDRATE METABOLISM

OF ETIOLATED CORN SEEDLINGS










By
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
August, 1960
















ACKNOWLEDGEMENT


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

graduate study.

The fullest credit is due my wife, who encouraged

the writer to undertake this study, and who worked so

untiringly in a determined effort to complete this step

in our lives.




























DEDICATION



to

my wife


Betty Louise Black














TA-.LE OF CONTENTS


Page

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

Plant materials
Preparation of enzyme extracts

EXPERIMENTAL PROCEDURE . . . . . . . 28

Studies on the Pentose Phosphate Pathway

Pentose disappearance
Chromatographic studies on pentose disappear-
ance
Assays for specific enzymes
Glucose-6-phosphate dehydrogenase
6-Phosphogluconate dehydrogenase

Studies on the Glycolytic Pathway

Phosphoglucoisomerase
6-Phosphofructokinase
Aldolase
Glyceraldehyde-3-phosphate dehydrogenase







Page


Phosphoglyceric kinase
Carboxylase
Phosphoglyceric mutase, enolase and pyruvic
kinase
Reagents used in these studies

RESULTS . . . . . . . . . 43

Studies on the Pentose Phosphate Pathway

Pentose disappearance
Chromatographic studies on pentose disappear-
ance
Glucose-6-phosphate dehydrogenase
6-Phosphogluconate dehydrogenase

Studies on the Glycolytic Pathway

Phosphoglucoisomerase
6-Phosphofructokinase
Aldolase
Glyceraldehyde-3-phosphate dehydrogenase
Phosphoglyceric kinase
Carboxylases
Phosphoglyceric mutase
Enolase
Pyruvic kinase

DISCUSSION . . . . . . . . . .70

SU.A.ARY A.ND COiJCLUSIONS . . . . . .. 79

I3 LIOGRAPHY .. .. . .. .... ... 82













LIST OF TABLES

Table Page

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

Figure PF

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


Vll













STATEMENT OF PROBLEM


Copious amounts of research have been directed at

attempting to elucidate the basic mechanism or mechan-

isms of action of plant growth-regulating materials. De-

spite the intensive and persistent efforts of research

workers to solve this problem, the basic mechanism or mech-

anisms of action of plant growth-regulators are not known

(56).

Since 2,4-dichlorophenoxyacetic acid (2,4-D) is one

of the earliest known synthetic plant growth-regulating

compounds, more research has been directed at attempting

to elucidate its basic mechanism of action than for any

other compound. A major area of research on the basic

mechanism of action of 2,4-D is its effects on plant meta-

bolism. Recently, Humphreys and Dugger (80, 81, 82) noted

that 2,4-D affected the catabolism of glucose in etiolated

corn seedlings (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).








glucose

A
V B
starch- ----glucose-6-phosphate- >-fructose-6-phosphate

C

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
.f 7-phosphate

ribulose-5- P
phosphate 1,3-diphosphoglycera
S \ 2 carbon
\ N / pool G

ribose-5- 3-phosphoglycerate
phosphate


2-phosphoglycerate
xyulose-5-
phosphate I


Glycolytic enzymes:

A. hexokinase
B. phosphoglucoisomerase
C. 6-phosphofructokinase
D. aldolase
E. triose isomerase
F. glyceraldehyde-3-
phosphate dehydrogenase
G. phosphoglyceric kinase
H. phosphoglyceric mutase
I. enolase
J. pyruvic kinase

Pentose phosphate pathway enzymes:


phospho-enol-pyruvate

J

pyruvate

krebs cycle


L. gluconolactonase
M. 6-phosphogluconate
dehydrogenase
N. phosphoriboisomerase
0. phosphoketopentoepimer-
ase
P. transketolase


K. glucose-6-phosphate
dehydrogenase


Figure 1. THE CATABOLISM OF GLUCOSE.


te








te


~ _~ ____ __________ ~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

new substances.









A complete review of the literature covering hundreds

of experiments on the effects of 2,4-D on plants demon-

strated several prominent factors. First, a large percent-

age of the data available was on the practical uses of 2,4-D

in agriculture. Second, many of the studies directed at

determining the effects of 2,4-D on plants were concerned

with morphological or anatomical effects produced by 2,4-D

at varying periods up to a year after application. Studies

of this type seem to be concerned with the result of 2,4-D's

action, but do not seem to be directly related to its in-

itial mechanism of action. Third, some short-time studies,

i.e. some as short as five minutes, showed that 2,4-D has

some effects on plants within a fairly short period of time.
Several workers have advanced the idea that to learn the

basic mechanism of 2,4-D's action, studies should begin soon

after its application to the plant. This approach seems

logical. Therefore, this review of literature will not cite

many research papers in which the data were collected long,

i.e. several weeks or months, after application of 2,4-D.

Fourth, although numerous hypotheses have been advanced con-

cerning the mode of action of 2,4-D, none have completely

withstood the intensive scrutiny of repeated and extended

research. Fifth, 2,4-D may have several modes of action,

and a search for the mode of action might be futile. Sixth,

several theories as to the mode of action of 2,4-D were









reasonably supported by research data and should be con-

sidered in a study of the possible mode or modes of action

of 2,4-D.

After considering the factors cited above, it was

decided to limit this review of literature to research

papers which deal with the probable initial mode or modes

of action of 2,4-D and several physiological responses to

2,4-D.


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

increased translocation.

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
2,4-D.

Cooke (33) noted that the uptake of calcium was en-
hanced initially by 2,4-D treatment, but that within 48

hours the uptake was decreased. Calcium has been reported

to be higher in leaves (165) and roots (164) of 2,4-D

treated plants, although Bass et al. (9) stated that six

days after treatment the calcium content of all tissues

of cranberry beans is lower than that of the controls.

The effect of calcium on cell wall elasticity and

plasticity is of particular interest due to the demonstra-

tion by several research groups that auxin may affect the

activity of pectin methylesterase (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

reported (9,33,160,165).


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

absorption.

The metabolism of nitrogen containing substances in

plants as affected by 2,4-D treatment has been studied

extensively. Although the results are not specific, several

general effects of 2,4-D treatment have been observed which

are reproducible. The total protein content of treated

tissue generally is increased (34,40,49,54,124,132,133,163,

172), but decreases have been noted in leaves (34,49,50),

while simultaneous protein increase in stems and roots of

the same plants resulted in a total increase in protein.

Galston and Kaur (55) fed labeled 2,4-D to etiolated pea

stem (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

bound electrolyte.

Studies on the effect of 2,4-D on amino acids have

been inconclusive, with increases (2,54,99,119,124) and

decreases (54,99,119) in free amino acids having been

reported. A partial explanation of this type of results

might be a stimulation of deaminating enzymes as re-

ported by Moewus (113). Akers and Fang (2) exposed
2,4-D treated beans to 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
(48,49,50,121,124).

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








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
theory (123,143).
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

later (166).
Both reducing and non-reducing sugars were lower in

stems of 2,4-D treated beans (132) and wild garlic (Allium

vineale L.) (94). Weller et al. (161) reported that
non-reducing sugars 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





23



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

this thesis.














,MATERIALS


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.





27




The nitrogen content of each extract was determined

by digesting 0.1 ml of the extract in 1.0 ml of sulfuric

acid, followed by Nesselerization. Therefore, the activ-

ity of each enzyme in each extract is presented on a

nitrogen or protein basis. The nitrogen concentration

was multiplied by 6.25 to obtain protein concentration.














EXPERIMENTAL PROCEDURE


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

28







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-

tulose, sedoheptulosan.

Hexoses were determined colorimetrically by the
method of Dische et al. (37). Ashwell (5) outlined the

procedure followed in these experiments. To determine
hexose, 1.0 ml of the supernatant fraction described

above was added to 4.95 ml of a mixture of six parts of

sulfuric acid to one part of water, cooled, then boiled





30



three minutes, cooled, 0.11 ml of cysteine hydrochloride

added, mixed and allowed to stand for two hours at room

temperature. The hexose concentration then was deter-

mined by obtaining the O.D. readings at 415 and 380 mu

and comparing these O.D. readings with those obtained

from known concentrations of the hexose, glucose.

Samples of known sugars were run in preliminary

experiments to test the reliability of the procedures

described above. In the range of sugar concentrations

used in these experiments, the procedures proved to be

quite reproducible. HIorecker and Smyroniotis (74) in-

dicated that fructose interfered with the pentose deter-

mination, but in these preliminary experiments it did

not.

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.





32



Assays for specific enzymes. The procedures described

in the remainder of this dissertation will be concerned

with assays for the activity of specific enzymes. In

assaying for each enzyme, preliminary experiments, not

reported in this dissertation, were run, in which the

limiting concentration of enzyme was determined for each

reaction. It was considered that the range of limiting

enzyme was obtained when the amount of enzyme could be

doubled to result in the amount of product being doubled

in a fixed time interval.

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

below:
4-
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:

G-6-P< >-F-6-P.


F-6-P gives about 65 percent of the color of free fructose

in this procedure (141). A typical reaction mixture

consisted of: 0.2 ml of Tris buffer (pH 9.0, 0.05 .);
0.2 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

F-6-P.

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+
GLYCERALDEHYDE-3- 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

below (137,155):

1. CH3* CO COOH- 3-CH3' CHO + CO2

2. C3 CO COOHI + CH3* CHO -< CH3- CO CHOH CH3+
002

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,

respectively, below:

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
extracts.
Enolase activity was assayed in the following reaction

mixture by utilizing the activity of a phosphatase in the

extracts, which would hydrolyze phospho-enol-pyruvate (PEP)








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

kinase activity.

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.













RESULTS


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.

43





















o a
X0
x k


-t V4 o0. anp
a6ueqo %





TU 9
/Pi 6uJ/saTown

TUJ 9
/saTown






GCI-' 01. anp
a6ueq3 ,/





TwJ 9
/u' 6w/saTown


TUJ 9
/saTown






C-tl4Z o0 anp
a6ueLq3 /





TW 9
/f,, 6w/saTown


TwU 9
/sa'own








**l.ua w1.eaI


C-


O*


co--


cnco





,--i
*


o [Y

- 4-4
N )





0
*-< --


0-4

-4-4


o*
O@(4
-4








01 (
+- I

i^ CM


CMO
\0 o



0 4
Oc,




,4-



wc,
,-- I


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


0
0)0

(L) l
XuLi


'j ^
C-1 I
r+^ ^
3

aaqun[; luawTzadx]


















ID Io (
IU) I U





0 ,-o


S10
* *













00 -n I





0 4 0











,3

3
*t IO *
II 10 I






























I I I 4J I
oII I
OO O3iO












0 +0










in 3L
OO3 CI&



I I





II I 1














* -0 *0
I-I~C'J


(0 0

0 E-




0 a 0

.3 C -








ac o
*o k 4














COO) > 0





O e.U
*,4 4-




00
.-4-H I





,0 a,


x ) < -0-






-on
o ( c )











O O0 c'
Ur 4-o 4-
S4-




00 *0
0 W
0 > 4 0





















C 4-'
U, -O
4- U CO -







%-POC > 4

:3 m 0P -4


04-' > *-H
0CO 4
CH C C10




;T -4
o0 a0





UOC ct
*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- ^


0

X

CY)
O




O
4-4
0
C
0
-H



4-'
C

U




c
0
u

-4

C




4,

r


c
E



4-)


4-'
ro






.A
n3

-I


0

4-'
4U
0(



4-'

o
4-3






0o
r0




*




*
*
*
N,







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

necessary.

As an indication of the completeness of the pro-

cedures used in assaying for sugars, the number of

umoles of carbon atoms utilized as pentose and used to

form heptulose and hexose was calculated from Table 1.

Table 2 contains this accounting of the umoles of car-

bon atoms utilized as pentose and found in heptulose and

hexose, following the 60-minute incubation period. In

the buffer treated extracts, from 53 to 99 percent of

the carbon atoms are accounted for in hexose and hep-

tulose, with an average of 79 percent. This 79 percent

of the carbon atoms compares well with the average of

84 percent in untreated tissue (Experiment 7). In the

2,4-D treated extracts, from 74 to 97 percent of the

carbon atoms were accounted for as hexose and heptulose,

with an average of 84 percent. This 84 percent accounting







C
0


U 0
-4G0


H C








C
w 3
LHEO

















+4- a
o o
, <




a0






0



0 0











00
o




o
0







+3
(o





a 3
a, -
0 0 0
S V 0
03 3
Ho



*r- a)


t-






0








W40








0-4
IOH



HO

c


<10













OI-
H






Co


LnO
o r_





0)0





CC


- 4 C') T n O \0 r-


om n-: r cc O co cr O
. . . " " d*





**

=NQ M NO ) O 0


C

48








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)


*
*
4,
c



t1-I
Cl,
W
H

rd-,



C
W
W-Q

E


ae
X 3
LU 2


O

*-4
x
C,)
Cr



c
*H
0


+-4




0






.-4
ca













-I-
0
o




+4
C




(3
0



.lq
C4-





U
3











0
4-)
X



F'r





0
c
*


a)









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




















TABLE 3


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

dehydrogenase.

The activity of glucose-6-phosphate dehydrogenase

was enhanced by 2,4-D treatment from 15.2 to 34.4 percent

over the activity in buffer treated extracts (Table 3).

Figure 2 includes a graph of typical data showing the

activity of glucose-6-phosphate dehydrogenase in cell-free

extracts from both 2,4-D and buffer treated tissue. 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

treatment.

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
































L.



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


,-I

0)

4-a,
Z. H

ULJ X
) 0J U) w-4
0 U C.
E




oo

oS a


U -- w)





4 U) L *

.o 1
t-Lt )









\nL.0 w C


I u"






) O8U
mO IU


















-O
0 C





















w H


0 *H
U 4J










x
OQ
700














L > .
Q C
U-l W u- *





.L, U-l
C3 I *
xO- J
10


















TABLE 4

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

treated extracts.

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

corn seedlings.

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.



















TABLE 5


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

observed.

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


Duffer
2,4-D

suffer
2,4-D

,uffer
2,4-D


0.63
0.55

0.86
0.30

0.55
C.48


12.7


7.0


12.7


-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



















TABLE 7


ACTIVITY OF ALDOLASE*


Experiment
Number


Treatment**


Klett Readings**""


% Decrease
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.




61






LU

CQ ZLU



N O O
< 0 -I

O0 -Q
O H (







_- -j 0 x






E -H 0 C





S(,- -4





C ~ c


a O. l


OC

0O >
-LU e


E oo
< <















F- -
C (m'X-(





r U *4
*C -P c *H










SC -C





"- 0 +
*-- i
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


















TABLE 8

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
timed intervals.


**See Table 1.







































0 0 0
O O O0
N -
N bwlo/9NiGV3 i.


(o' )t I 1 dO
(nwOt72)AllSN3a -VDI.LdO


64




*,4


n "
00 X


SE *-4
0


U )





0 = *-3 4J
-EU O


O C,-I
.o -- -
.C: r-4 X :
HOOO
u U) U U







o u







0 (D F -4
O 0















.o co H <
-- H H -- 0 -
4 I C o








S0 H0









.CO 0 0
0. 0 40C 04
3~o +-, ("l >





S0 3CO (



00 -0 *
i- a- .-



o -- L *4- Q

) H0
0 0r0 *0
E a) 0



0 .4 (D C C:
aO 4-
\~ ~ 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-

glyceric kinase.

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-

ments.

In these studies with acetone powder extracts, a high

endogenous phosphatase activity was noted in the reaction

mixtures minus ADP which made measurement of this enzyme

difficult. Inclusion of ADP in the reaction mixture did

not increase the pyruvate formation above the endogenous




















TALLE 9


ACTIVITY OF ENOLASE*


Experiment Treatment** Klett readings
Number per mg protein


26 Buffer 20
2,4-D 20

27 Buffer 36
2,4-D 37

28 Buffer 27
2,4-D 26


*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-

phatase activity.

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

were inconclusive.














DISCUSSION:


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

phosphorylation (90).

Consideration of this reversal of the effects of

2,4-D by soaking the treated seedlings raises a question

concerning another portion of this study. Since soaking

intact seedlings will reverse 2,4-D effects, why doesn't

dialysis of the extracts have similar effects? In this

connection it should be noted that glyceraldehyde-3-

phosphate dehydrogenase was studied in acetone powder

extracts which were not dialyzed. Thus, the reversal of

the effects of 2,4-D by soaking intact tissue was noted

on both dialyzed cell-free extracts and undialyzed acetone

powder extracts.

The results of this study support the observations by

Humphreys and Dugger (80,81,82) concerning the catabolism

of glucose in intact corn roots. Their in 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-

sis.

In this series of experiments, the enzymes of both

glycolysis and the pentose phosphate pathway were present

in the extracts. Even though previous in 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.




78



Thus, experimental data support the theory that

2,4-D could affect the metabolic control mechanisms. The

elucidation of this mechanism will be dependent upon further

research employing such research techniques as those of

Chance and IIess (30), whereby small differences can be

measured accurately in short-time intervals employing

intact tissues.














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

seedlings.

Three-day-old etiolated corn seedlings were divided
-2
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

conclusions:

1. Glycolytic enzymes and the enzymes of the

pentose phosphate pathway were present in extracts from

roots of both untreated and 2,4-D treated corn seedlings.

2. The activities of 6-phosphofructokinase, aldolase

and glyceraldehyde-3-phosphate dehydrogenase were de-

creased in extracts from 2,4-D treated tissue, while the

activities of phosphoglucoisomerase, phosphoglyceric

kinase and enolase were not affected. Studies of phospho-

glyceric mutase were not conclusive.

3. The activities of glucose-6-phosphate dehydro-

genase and of 6-phosphogluconate dehydrogenase were

enhanced in extracts from 2,4-D treated tissue.

4. In extracts from 2,4-D treated tissue, there

was an increased utilization of R-5-P and an increased

formation of heptulose and hexose.

5. These 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

phosphate pathway.




81


6. A theory is presented and discussed which

proposes that 2,4-D in some manner effects the normal

metabolic control mechanisms of intact cells.















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.
1955.

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

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

13. Berger, J. and G. S. Avery, Jr. Glutamic and
isocitric acid dehydrogenases in the 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.
46:279-290. 1953.

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.
113:482-484. 1952.

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:
533-536. 1952.

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.
107:332-343. 1946.

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








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.
1959.

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

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

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

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

41. Erickson, L. C., R. T. Wedding and B. L. Brannaman.
Influence of pIH on 2,4-dichlorophenoxyacetic acid
and acetic acid activity in Chlorella. Plant Physiol.
30:69-74. 1955.

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.
35:405-408. 1960.

43. Fang, S. C. and J. S. Butts. Studies in plant meta-
bolism. IV. Comparative effects of 2,4-dichloro-
phenoxyacetic acid and other plant growth regulators
on phosphorus metabolism in bean plants. Plant Physiol.
29:365-368. 1954.

44. 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.
113:322-333. 1952.

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.
959.

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

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

58. Gatt, S. and E. Racker. Regulatory mechanisms in
carbohydrate metabolism. 1I. Pasteur effect in
reconstructed systems. J. Biol. Chem. 234:1024-
1028. 1959.








59. Gibbs, A. Triosephosphate dehydrogenase and glucose-
6-phosphate dehydrogenase in the pea plant. Nature
170:164. 1952.

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.
57:421-436. 1955.

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.
1956.

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-
3055. 1959.

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.
1947.

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
Suppl.:xxii. 1956.

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.
32:136-140. 1957.

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

81. Humphreys, T. E. and W. 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.
73:139-143. 1957.

84. Johnson, E. J. and A. R. Colmer. Further studies
on the mode of action of 2,4-dichlorophenoxyacetic
acid on Azotobacter 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.
1958.

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.
1949.

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

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

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

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
118:627-628. 1953.

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.
35:126-128. 1960.









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-
150. 1954.

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

115. Neely, W. B., C. D. Ball, C. L. Hamner and 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:
118. 1950.










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-
623. 1960.

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

122. Rasch, E., H. Swift and R. 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.
1954.

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




University of Florida Home Page
© 2004 - 2010 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Last updated October 10, 2010 - - mvs