A STUDY OF THE LACTIC AND MALIC OXIDATIVE ENZYMES OF TOBACCO SEEDLINGS GROWN IN ASEPTIC CULTURE

72 Pages·02.729 MB·English

by  RIDEN, JOSEPH R., JR

DOCTORAL DISSERTATION SERIES
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The Pennsylvania State College
The Graduate School
Department of Agricultural and Biological Chemistry
A Study of the Lactic and Malic
Oxidative Enzymes
of Tobacco Seedlings Grown in
Aseptic Culture
A Dissertation
by
Joseph R. Riden, Jr.
Submitted in partial fulfillment
of the requirements
for the degree of
Doctor of Philosophy
June 19^1
Approved: ^ ^
PrnfesS'orf of Phytochemistry
Head, Department of Agri-
cultural and Biological
Chemistry
AC KN 0 VVLEDGEMENT
The writer wishes to express his appreciation
to Dr. C. 0. Jensen for having suggested this problem and
for his advice and criticism during the course of this
thesis and its preparation; and to Dr. R. A. Steinberg,
Department of Agriculture, Beltsville, Maryland, for
demonstrating the technique of growing tobacco plants in
aseptic culture.
TABLE OF CONTENTS
Page
I INTRODUCTION ...................................... 1
II REVIEW OF LITERATURE..................... 3
A. Plant Oxidative Enzymes ...................... 3
B. Methods for the Determination of
Oxidative Systems ........................... 10
III EXPERIMENTAL
A. Development of Method
1. Sterile Culture of Tobacco Plants . . 13
2. Preparation of Plant Tissue for
Enzyme Studies................« . . . 15
3. Details of Methods Used to
Determine Oxidative Enzymes .......... 21
B. Measurement of Plant Enzymes
1. The Lactic Enzyme
a. Cofactors Necessary for
Activation................... 21j.
b. pH S t u d i e s ................... 26
c. Determination of Carbon Dioxide
Produced..................... 28
d. Cyanide Inhibition Study • . • • 29
e. Optimum Substrate Studies • • . • 32
f. Triphenyltetrazolium Chloride
Studies . . • • • • • 32
g. Methylene Blue Studies on
Acetone Extracted Tissue . . . . 36
h. The Effect of Sucrose Upon
Lactic Oxidase ................ 38
Page
i. Lactic Oxidase Content of
Etiolated Tissue ............... 39
j. Purification of Lactic Oxidase . I4J.
k. Identification of an End Product L^2
2. The Malic Enzyme...................... 2+3
a. Cofactors Necessary for
Activation...................... lj.3
b. pH S t u d i e s ...................... 2+7
c. Determination of Carbon
Dioxide Produced............... 2+7
d. Triphenyltetrazolium Chloride
Studies.......................... 5>0
e. Methylene Blue Studies on Acetone
Extracted T i s s u e ............. . 51
IV DISCUSSION OP R E S U L T S .......................... $$
V SUMMARY AND CONCLUSIONS ........................ 60
VI BIBLIOGRAPHY..................................... 62
A
I INTRODUCTION
"When distressed by the thought that in your own
particular branch of biochemistry progress is slow,
significant facts are few, and confusion is rampant, you
will be consoled to learn that some other fields of
biochemistry are in an even more primitive state of
development*"
" .... In view of the present state of knowledge
of plant respiratory enzymes it would be unwarranted to
accept such an assumption without proof."
The first statement was made by Lardy (27) in
1951 while reviewing the latest in a series of volumes on
enzymology. Maxwell (36) was speaking about the presence
of cytochrome oxidase in corn embryos when he published
the latter statement in 19^0. This thesis was undertaken
in hopes of making a contribution to the now meager
knowledge of plant enzymes and the methods used to study
them.
When a method appeared in the literature for
growing tobacco plants in aseptic culture, the technique
was adapted for this study of tobacco enzymes. By using
sterile tobacco plants, the contaminating enzymes of
bacteria, molds, fungi and other living matter found on
tobacco grown under field conditions can be eliminated.
By the proper handling of sterile plant tissue, we may be
certain that we are studying only the enzymes which are
2
found in the green tobacco plant.
Cigar leaf tobacco is an important crop in
Pennsylvania agriculture. The tobacco must be cured or
fermented before it is usable, such processes probably
being caused by enzymes. These enzymes may be of bacterial
or plant origin. Jensen and Parmele (23) have studied the
fermentation of cigar-type tobacco and have isolated and
identified seven species of bacteria from fermenting
tobacco. Reid, McKinstry, and Haley (5l, 5>2) have also
made a study of the bacterial flora of fermenting tobacco,
and have shown that more than one billion bacteria per
gram may exist in sweating tobacco. The contributions of
bacterial enzymes to the changes taking place in fermenting
tobacco are undoubtedly important. However there is not
sufficient evidence to indicate that plant enzymes play
no part in the process.
The relation of enzymes of the tobacco plant
and enzymes of microorganisms to the curing of tobacco is
largely unknown. Before such knowledge is produced more
information concerning the enzymes of tobacco itself and
a study of the microflora must be available.
3
II REVIEW OP THE LITERATURE
A. Plant Oxidative Enzymes. The oxidative enzymes of
higher plants have not had the thorough investigation that
has been given to the oxidative systems of animals and
bacteria. A partial explanation for this may be the lack
of a common oxidative system such as that found in all
animals. The literature on plant enzymes is composed of
many small pieces of information on the variation of
oxidative systems between species of plants between differ­
ent parts of the same plant, and even between the same plant
at various stages of growth. For example, Tolbert and
Burris (61) reported that the leaves, but not the roots, of
the mature tobacco plant contain glycolic oxidase, which is
not present in etiolated tissue and which is not stable in
young plants. Also, Albaum and Eichel (2) found that
respiration in oat seedlings is mediated by the cytochrome
system for the first 72 hours, but after that period is not
inhibited by any of the cytochrome system inhibitors. As
a result of this variation in the enzyme composition of
plants, a review of the literature provides few general­
izations and many inconclusive statements regarding plant
oxidative enzymes.
The oxidative enzymes in plants may be classified
either as dehydrogenases, which remove hydrogen atoms from
substrates and pass them along a chain of carriers to
atmospheric oxygen, or as oxidases which directly unite the
1*
substrate's hydrogen or the substrate itself with oxygen.
A terminal oxidase is an oxidase which accepts electrons
from the dehydrogenase carrier chain, and reduces molecular
oxygen, thereby completing the system. In a study of the
electron carriers of plant dehydrogenase systems, Whatley
(68) found only traces of diphosphopyridine nucleotide (DPN,
Coenzyme I) in green leaves, although 20 ;ug. of triphos-
phopyridine nucleotide (TPN, Coenzyme II) per gram of dry
tissue weight was present. Lockhart (31) found flavoprotein
in peas, beans and white potatoes, although it was stable
only four days. Bach (4) reported that garden vegetables
were rich in dehydrogenases, but contained very little DPN.
In studying the dehydrogenases of plant tissues, it was
necessary to add DPN to activate the dehydrogenases.
Berger and Avery (7) have made a study of malic
dehydrogenase in the avena coleoptile, using Thunberg
techniques with thionin as the hydrogen acceptor. The
enzyme was purified by ammonium sulfate fractionation and
activity was obtained only after DPN and flavoprotein were
added to the system. An accumulation of the end-product,
oxaloacetic acid, inhibited the system, but could be
removed by binding it with cyanide. The addition of various
non-heavy metallic ions had no effect upon the system.
Thionin was used in this study and prevented gaining any
knowledge of the nature of the terminal oxidase. I'alic
dehydrogenase has been found in corn embryos (2lj.), spinach
leaves (69), pollen (5»5>) and parsley roots (65).
$
Succinic dehydrogenase has been round in wheat
term by Goddard (20) who reported that it was destroyed by
acetone precipitation of the tissue. In spinach leaves,
Bonner and Wildman (11) found it rapidly destroyed after
injury to the leaves. This instability of succinic dehydro­
genase may well be the reason that Berger and Avery (7)
were not able to detect it in oat seedlings. This dehydro­
genase has been found in corn embryos by Jensen, Sacks and
Baldauski (2lj.). A detailed discussion of this enzyme has
been given by Stauffer (55).
Mathews and Vennesland (33) examined the action of
formic dehydrogenase which they isolated from green pea
seeds. They found DPN was necessary for the reaction and
followed its reduction spectcphotometrically. The formic
dehydrogenase reduced the DPN, while another enzyme
catalyzed the oxidation of DPN by methylene blue. The plant
formic dehydrogenase received no direct stimulation from the
addition of the adenosine phosphates; this being in contrast
to animal formic dehydrogenase. Formic dehydrogenase has
been found in many plant seeds.
Eerger and Avery (8) have studied the glutamic
dehydrogenase of avena coleoptiles. They have shown that
it requires DPN for its action and had an optimum pH from
6.5 to 8.7. The enzyme was inhibited by 0.001 molar copper -
and cobalt solutions. Glutamic dehydrogenase also appears
in spinach leaves (11).
Isocitric dehydrogenase has been found in the avena