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EFFECT OF CERTAIN NUTRIENT
CONDITIONS O\' ACTIVITY
OF OXIOASE AND CATALASE

Thesis for Degree of M. S.
Boyce De‘X/itt 326“

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Effect of Certain Nutrient
Conditions on Activity
of Oxidaee and
Cetalaee

THESIS

Submitted to the Faculty of the Michigan State
College of agriculture and Applied Science
in partial fulfillment of the require-
ments for the degree of
Master of Science

1}.
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/ By
, - Boyce DeWitt E2911
June 1926

THESIS

- 1 -

INTRODUCTION

Enzymes have long been known to be important
constituents of plant cells. During recent years more
and more importance has been attributed to their activities.
Certain plant physiologists have termed the enzyme activity
of a plant as 'the strategic center of vital phenomena".
This might lead one to suppose that when any factor, such
as disease or nutrient supply, operates to retard or in-
crease the growth of a plant the direct results may
possibly be due to a change in the enzyme content.

The available experimental evidence shows that
the active enzyme content of the plant is not always a
fixed constant but subject to variation through the in-
fluence of such conditions as disease, changes in the re-
action of the tissue fluids, etc. In view of the fact
that nutrient substances, when properly applied, increase
the tone and growth response of the plant, it may be
supposed that they too are effective by causing favorable
stimulation of the essential enzymes. Since little or no
work has been done in determining the effect of plant
nutrients on enzyme activity in herbaceous vegatation, this
investigation was undertaken as a study of that relation-

ship 9

REVIEW‘OF LITERATURE
In reviewing the literature the work of Sullivan

(21) stands out as bearing directly on the effect of various

98090

salts on enzyme activity. He found that potassium sul~
phate even in dilute solutions had a retarding effect

on the oxidizing action of wheat roots, while sodium
nitrate and mixtures of Ca H4IP o4I2, Na n 03 and K2 s 04
had an accelerating effect. In testing the action of
these salts in solutions with concentrations of 100 parts
per million of 2205, N H5, and 12 0, used singly and also
in combination, on commercial malt diastase in starch paste
he observed that GaH4IPO4I2 increased the diastatic activity
of the enzyme from 8 to 16%; that a mixture of equal parts
of 0a £49003. Ba Hos and K2 304 increased the diastatic
activity from 6 to 16%. While Kg 304 alone uniformly re-
tarded the enzymatic action. Na N03 alone made little
change in the rate of conversion of the starch to reducing
sugar. Plants growing in solutions containing 100 parts
per million of either 2205, M3 or K80, or a mixture of
equal parts of these and 200 mgm. of starch paste, had the
power to convert the starch to sugar. As a rule, the
diastatic activity was retarded by K2304 and Ca B4IPO4IZ
but was increased by either Na N025 or a mixture of the
three salts.

Heinicke (18) found that nitrogenous fertilizers
increased catalase activity in leaves from apple trees
growing in sod, also that leaves from trees growing in
sod showed less activity than leaves from trees under

cultivation, and leaves'from trees growing in sandy soil

less than those growing in clay loam. In general his
work showed that the presence of growth producing sub-
stances (nutrients) favors catalase activity while sub-
stances that tend to inhibit vegetative growth have a re-
tarding influence on its activity. As a result he sugu
gested the use of the catalase test as an indicator of the
physiological responses of apple tree tissue to cultural
treatment or conditions. He (13) obtained similar results
from the bark of dormant trees. Auchter (4) working with
trees and shrubs to which nitrate had been applied to one
side found increased activity only in the foliage of the
side on which the fertilizer was applied.

Weiss and Harvey (25) working on potatoes in-
fested with potato wart disease found that the wart tissue
had greater catalase activity and also a greater pH con-
centration than the healthy tissue. With other plants the
diseased tissue showed increased activity in every case
but the pH concentration was less in the diseased tissue.
In all plants studied the pH values were greater than
pH 7.0.

Bunsel (7) concluded that the oxidase content
was low in a normal plant but increased greatly if the
growth was interfered with by drouth, excessive moisture,
diseases or any other factor that tends to retard growth.
With the potato plant grown normally, he found that the
young shoots showed great oxidase activity which later be-
gan decreasing in amount and continued falling until the

-4-

fortieth day after planting, after which time it began.in~
creasing again as the plant came to maturity.

Moore and Whitley (16) demonstrated that the
distribution of oxidases varies widely in the same plant,
being present in the largest quantity in the region con-
taining the greatest abundance of "respiratory vessels".
Woods (24) found that the oxidase content of tobacco
suffering from mosaic was greater in the diseased areas
than in normal ones: the oxidase content being inversely
proportional to the chlorophyll present as Judged by color.
Rose (19) has shown that the oxidase content of apple tree
bark affected with Illinois canker is greater than in
normal bark.

Cook and Taubenhaus (8) found oxidase abundant in
young fruits, and gradually decreasing as the fruit ripen-
ed. This loss of enzyme activity seemed to be accompanied
by increased susceptibility of the fruit to disease. The
authors suggested that the oxidase content of the fruit
might be directly related to disease resistance through
the production of tannin by the oxidase. Appleman (1)
found a great varietal difference in the oxidase content
of potato tubers. The.Mc Carmick variety (grown in Mary-
land) had an oxidase activity four times greater than that
of Carmen No. 1 (grown in New York). When specimens of
the two varieties had been.kept in storage under the same
conditions for a period of two months. Bunzel (6) found

that large healthy leaves contained less oxidase than small

-5-

healthy leaves and that leaves collected at sundown showed
much greater oxidase activity than leaves collected at sun.
rise. If any normal function (such as failure to develop
seeds in the normal way) was inhibited in any manner oxidase
activity was increased. He also discovered a general
parallelism between oxidase activity and green color of the
green part of the plant (beets). True and Stockberger (82)
working with the opium poppy plant (Papaver somniferum)
found an increased activity from base to summit of plant
with greatest activity in floral structures, capsules and
all actively growing parts.

The available experimental evidence does not
warrant a definite conclusion regarding the relation of
nutrient substances or other growth-promoting factors to
the activity of oxidase and catalase. Some investigations,
particularly those of’Heinicke and huchter (previously
cited) yielded data showing that plants whose growth was
stimulated by certain nutrients showed increased catalase
content, while other investigations have resulted in the
accumulation of facts which show as conclusively that the
activity of both oxidase and catalase was increased when
normal growth was in any way prevented or interrupted. In
the work to be reported herein herbaceous plants, free from
disease, grown in a great variety of nutrient and environ-
mental conditions were used. Numerous determinations were
made and the data obtained therefrom.may serve as a con-

tribution towards the solution of this important problem.

 

 

 

 

 

- 5 -

KETHOD

Data were taken from four different experiments
(described later), essentially the same methods for measur-
ing enzymic activity having been used in each instance.

The manner of the preparation of the samples was similar to
that used by Heinicke (12) for catalase determinations.

By means of a leaf punch two disks per leaf were taken from
two leaves at the same level on the opposite sides of each
of several plants. The disks, one centimeter in diameter,
were taken from the opposite sides of the midrib and about
two-thirds the way to the tip. The leaves from which
samples were taken were Just reaching vegetative maturity.
The disks were weighed immediately and mixed with an equal
amount of dry calcium carbonate and enough water to cause
the calcium carbonate to spread evenly over the material.
After the disks were thoroughly coated with the calcium
wash they were ground gently in a mortar until a uniform
creamy mixture was obtained. Distilled water was then
added so that one gram of the fresh tissue was suspended

in 25 cc. The solution was then placed in a tightly
stoppered bottle and kept until ready for use.

‘In testing enzyme activity use was made of Harvey's
modification of the simplified Bunzell oxidase apparatus (11)
suspended in a ‘DeKhotinsky thermostatic bath. The temperap
ture of the bath was held constant at 36°C; .fluetuations not
exceeding 0.100. Wire baskets were used to hold the oxidase

tubes while immersed in the bath and to allow uniform shak-

 

 

 

Fig. 1. Apparatus Used for Enzyme Determinations

-8-

ing to be accomplished by means of a-mechanical shaker. The
shaker was driven by the motor used to run the stirrer of the
bath (Fig. l). The rate of shaking was such as to cause
the solution to flow from one end of the tube to the other
40 times per minute. In making a determination of oxidase
activity, the suspension of macerated tissue was first
thoroughly mixed by shaking the bottle gently and then taking
2 cc. of the content and placing this in the short arm of
the reaction tube. In the other arm of the tube 5 cc. of a
fresh one percent pyrogallol solution were introduced. The
alkali tube containing 1 c.c. of normal solium hydroxide
was put in place, the manometer adjusted and the tube placed
in.the bath. .As soon as the contents came to the temperature
of the bath, the tube was closed, shaking started and con-
tinued for one hour. The reading of the manometer at the
end of one hour was taken as a measure of oxidase activity.
In case positive pressure was developed at the beginning
correction for this was made at the end.

For catalase the same sample was used as for
oxidase, but diluted to give one gram of fresh tissue to
100 c.c. of water. Catalase determinations were then made
as for oxidase except that 5 c.c. of 3 per cent hydrogen
peroxide, previously neutralized with sodium carbonate,
were substituted for the pyrogallol solution and the read-
ings of the manometer taken at the end of one, two and three
minutes. For determining the catalase activity in Experiment
4 the water displacement method as described by Heinieke (13)

-9-

was used and the leveling tube adjusted so that a constant
pressure of one atmosphere was maintained. The amounts of
water displaced at the end of one, two and three minutes
‘were taken as measures of the activity of the enzyme.
Preliminary tests showed that from the time of
preparation of the sample oxidase activity increased for
about six hours, when the sample was kept at room temperature.
.After reaching a.maximum there followed a period of stability
lasting 10 to 18 hours, after which there was a slow decline.
Data showing this are presented in Table 1.

Table l. -- Effect of Time on the Oxidase Activity
of Prepared Samples of Lettuce Tissue.

 

Time,fiafter preparation’ 6n ndase Activity in.l hr.

 

 

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Consequently, the oxidase determinations were
made between the sixth and eighteenth hours following the
preparation of the samples. Catalase is less unstable than
oxidase and retains its original activity for a much longer
time. This being true the determinations of catalase were
made the day following the oxidase tests. .All determinations
were run in duplicate and discarded and repeated, when not
in close agreement. All results shown in Tables are averages

of the duplicates.

-10..
EXPERIMENTAL

Win-m 110- ,
The soils used in this experiment were (1) muck

(2) a mixture of’muck and sand, and (5) a good greenhouse
potting soil. The lime requiredment of the muck was 34,200
lbs. calcium oxide, and that of the mixed soil 15,000 lbs.
calcium oxide per acre according to the Jones method of
determination (14). The potting soil was neutral to litmus.
A.basio treatment of commercial sodium nitrate and potassium
chloride (the former at 1000 lbs. per acre, the latter at
2500 lbs. per acre) was given to both the muck and the mixed
soil several weeks before the plants were set and the ex-
periment started. Hydrated lime and commercial acid
phosphate were used in various amounts and proportions as
shown in Tables 2 to 4. These treatments were also applied
at the same time, as the basic applications mentioned
above, the soil being kept moist during the interval.
Lettuce of the Grand Rapids Forcing variety was
used. The seeds were germinated in flats of soil, pricked
off once, and later carefully selected for uniformity of
size and vigor and transplanted to the experimental flats.
Six plants were grown in each flat, the flat being 12 x 21 x 3
inches, two flats being used per treatment. The flats were
placed on the boards of the bottom of a greenhouse bench in
order to confine the roots. Environmental conditions were

kept optimum for the growth of lettuce and uniform for

- 11 -

all the flats. Watering was watched closely and the soil
moisture held as near as possible at the apparent Optimum
for each soil. The duration of the experiment was 80
days. The data are set forth in tables 2 to 4 inclusive.
The data in Table 2 show little if anything re-
garding catalase, while decreases in oxidase activity where
the plants were treated are quite evident. The differences
for lots 2, 3 and d as compared with lot 1 were 20, 8 and
12 percent respectively. Clearly, positive correlation
between the growth effect of the soil treatments and en-
zyme activity is wholly lacking.
As seen in Table 3 the soil treatments fall into
three series: increasingly large applications of both
lime and acid phosphate, increasingly large applications of
lime with amount of phosphate constant, gradually larger
amounts of line with no phosphate. The results show that
neither the lime nor the phosphate affected oxidase
activity very significantly. In Series 1 the plants were
not significantly altered in size as the amounts of lime
and acid phosphate increased, and the oxidase activity re-
mained essentially the same as compared with the check.
In Series 2 the same thing prevailed, while in Series 5
there is some indication that the additions of lime in-
creased oxidase activity; decreasing at the same time the
growth of the plants. This is in agreement with Bunzell
(6) (7) who found that anything which checked normal
growth tended to accelerate the action of oxidase.

-12..

 

 

 

 

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- 15 -

As regards catalase the data are somewhat definite.
-Where growth was reduced as in Series 3 (lime only) the
activity of the enzyme was increased, while in Series 1
(lime and acid phosphate) the size remained practically the
same with a decrease in catalase.

As may be observed from Table 4 there were two
series of soil treatments: increasingly large applications
of lime with the amount of phOSphate the same; no phOSphate
with larger and larger amounts of lime. In both series the
activity of oxidase and catalase was increased over that
of the check by the soil treatments. In Series 1 the size
of the plants was not consistently influenced in either
direction. In Series 2 growth was gradually diminished.
Experiment 2 -- Lettuce and Radish.

In this experiment two soils were used (1) a

 

good soil consisting of five parts loam and one part sandy
gravel (Series 1), and (2) a poor soil consisting of five
parts sandy gravel and one part loam (Series 2). Fertilizer
treatments were applied as shown in Tables 5 and 6, one gram
of each nutrient substance (c.p.) being applied per pot.
Where cumulative doses were applied one-fourth of the total
amount was aoplied at the beginning and the remaining one-
‘fourth portions at successive intervals during the experiment.
The lettuce grown was of the Grand Rapids Forcing
variety. The seedlings were treated as in Experiment 1
but were grown in six inch pots instead of in flats,

a single plant per pot. Twenty pots were used for

-16-

each treatment. The pots were filled uniformly with the
soil and kept plunged in sand on a greenhouse bench. The
pots were systematically distributed to offset any place
effect and to give equal exposure to the individual plants
of each series. Environmental conditions were similar to
those in Experiment 1. The plants were grown for 94 days,
or from.Hovember 7 to February 9.

The radish grown was the Scarlet Globe variety.
The seed was planted in flats on December 15 and trans-
planted to six inch pots January 15. The soil was the
poor soil (Series 2) mentioned above,and the fertilizer
treatments those shown in Table 6. Environmental condi-
tions were similar to those surrounding the lettuce plants.
The plants were grown for 37 days.

.A study of Table 6 shows that the size of the
plants was increased by the nutrient treatments in.both
series except in lots 36 and 43 where potassium was used.
Changes in oxidase activity were not significant in either
series, and did not follow the variations in the develop-
ment of the plants. The same was true regarding catalase;
the determinations varying considerably but not consistently
nor at all positively with the increases in the growth of
the plants from lot to lot.

Table 6 shows that while the size of the tops of
the radish plants was affected by the size of the fertilizer

treatments the oxidase content remained unchanged except

-17-

 

 

 

 

 

 

 

 

 

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in the nitrogen lot where the oxidase activity was lessened.
rho catalase activity was increased slightly over the check
by nitrogen and by potassium when applied alone, but

lowered when both were added together and also lowered when
phosphorous was added. Qhere was no positive correlation
between size of plants and catalase activity, but rather a

tendency towards negative correlation.

n -- L d 8 h
Spinach plants of the Savoy type and lettuce

plants of the Grand Rapids Forcing variety were used. The
seedlings were grown from.select seeds and transplanted

to six inch pots on Feb. 24, 1926. One plant was grown in
each pot with seven plants for each series or treatment.
the length of the growth period was 40 days.

The soil was a.mixture, consisting of two-thirds
medium coarse sand and one-third fine sandy loam. One-
half of the total amount of each fertiliser treatment was
mixed in the dry state into the soils of the several series
Just before potting; the remainder was added in solution
on March 12, sixteen days after potting. The nutrient
salts were all of c.p. quality. The pots were filled
uniformly with the soil and kept plunged in sand on a
greenhouse bench. Ihe sand about them was kept moist.
Water was applied in exactly equal quantities per pot dur-
ing the period of growth. The pots were systematically
distributed as in Experiment 2. Development within each

Check )
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- 20 -

 

 

 

 

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series was uniform. The differences between the plants of
the various lots due to the treatment were very definite
and consistent (see Figs. 2 and 3). The results are given
in Table 7.

Table 7 shows consistent increases in the size of
the plants, following the nutrient treatments. The activity
of oxidase was not materially affected nor different within
the several lots. In general, the catalase content was
greater where the nutrients were applied, but the increases
do not consistently follow the size of the plants nor
were they correlated in detail with changes in growth.

Table 8 shows that the nutrient treatments re-
sulted in increased growth of the plants. Oxidase activity
did not vary with increases in size of plants but rather in
the opposite direction. ‘Again, as with the spinach plants
of this experiment catalase activity was greater in general
in the fertilized plants, but the variations are not at
all closely parallel.

Experigegfi 5 -- Lgttugg.
In this experiment the effect on enzyme activity

of lengthening and of shortening the normal day in con.
Junction with nutrient treatments was studied. Grand
Rapids Forcing lettuce plants were grown singly in six-
inch pots of fine sand low in fertility. Various nutrient
substances were applied to the soil and the periods of

illumination of the plants controlled. Three series varying

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in the amount of illumination given were used. Series 1
(short day) was given daylight illumination from.9 Adi.

to 3 2.3!. and covered with specially built light-tight
boxes the rest of the time. These boxes were 10 feet long,
5 feet wide and 3 feet high, with light-tight ventilators
arranged in each end so that circulation of the air within
might take place. Series 2 consisted of check plants grown
in normal daylight (Nov. 5 to Jan. 3). and Series 5 (long
day) received illumination from daylight in the morning
until 11 P. M. The extra illumination of these plants was
accomplished by means of Hades light bulbs (1000 Watts each)
socketed in porcelain enameled steel dome reflectors sue»
pendedat a height of 4.5 ft. above the bench. Three of
these bulbs, evenly spaced apart were used over a bench

81 ft. long and 5 ft. wide, the pots containing the plants
being distributed so as to receive equal illumination, and
being systematically shifted about each week. These plants
were grown in the winter months (Nov. 5 to Jan. 3) so the
daily period of illumination'was lengthened about 5 hours.
The periods of illumination of the different series were
approximately 6, 10 and 16 hours respectively. The pots
were kept plunged in sand on a greenhouse bench and moisture
and temperature conditions kept favorable for growth. From
15 to 22 plants were used for each treatment in the different
series. and plant nutrients supplied as shown in Tables 9 and

10, one gram of c.p. material being added to each pot.

In each series the fertilized plants were larger
than the check in the same series. In Table 9, Series 1
there is no change in the oxidase activity with the_differ-
ent treatments. Series 2 consisted of a.normal day check
only, while in Series 3 fertilizer treatments and growth
were accompanied by increased oxidase activity, and also
by some increases in catalase activity. In Table 10
Series 1 there was a decrease in oxidase activity with the
fertilizer treatments and growth. The same is true in
Series 3 with the exception of Lot 69 which showed increas-
ed oxidase activity and lowered catalase activity in com.
parison to the other lots in same series. The last applies.
tion of fertilizer was made ten days before the samples were
taken, so the alteration of the enzyme activity was perhaps
not due to this application. The catalase of the other
lots in Series 3 was not materially affected; in Lot 71
where nitrogen and phosphorous was applied cumulatively the
catalase activity was slightly higher.

Differences in the length of day had but slight
effect on the activity of catalase, while with the short
day there was a tendency towards increased oxidase activity.

With oxidase the samples taken at different times
during the growth period showed little difference, four
out of the nine lots showed slightly greater activity on
December 4 than they did on December 81, the other five
showed slightly greater activity on December 21. The

catalase activity was greater in every case on the latter

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-28-

date. It is also noteworthy that in the plants of this ex.
periment oxidase activity on the whole was far greater
than in the plants of any previous experiment conducted
under different conditions.

DISCUSSION

The obvious tendency in the results that have
been presented is for greater activity of the enzymes to
be associated with diminished growth and size of the plants.
In order to test, and confirm or else disprove this ob-
servation coefficients of correlation were determined
respecting average green weights of the tops of the plants
of all the lots of all experiments and the activity of both
oxidase and catalase, using the manometer readings at 5

minutes for catalase. The Pearsonian formulaSle was
n cgd})

used for calculating the.cosfficient of correlation, and
the formula - $.6745 for obtaining their probable
errors. The results are shown in Table 11.

Table 11 shows clearly that correlation existed
between the average size of the tops of all plants of the
several experiments and enzyme content of their tissues,
that the correlation was highly significant with both
oxidase and catalase,-being"nearly~perfectvreepecting—the

—latter. and further that the correlation was negative in
character. This means that reduced activity of the en~
zymes (oxidase and catalase) was associated with greater

growth of the plants. In addition it tends logically to

 

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the conclusion that since nutrient substances resulted in
increased growth of the plants their effect relative to
‘these enzymes was to diminish their content and activity.
. This reasoning which would establish the following chain
of antecedents: increased nutrients -- decreased oxidase
and catalase activity -- increased growth, assumes, of
course as a premise that growth is directly subject to
changes in the amount or properties of the enzymes under
consideration. It also, neglects taking account of any
effect of nutrient substances which may be exerted other-
wise than indirectly through affecting these enzymes.

It is true that Heinicke (12) (13 ) and also
Auchter (4) have reported finding increased catalase
activity associated with greater vigor of fertilized trees
and shrubs. Perhaps the facts are not the same for woody
plants as for herbaceous ones. In an effort to duplicate
the results of the above mentioned workers determinations
of catalase activity were made during the summer of 1925
(July 15) on apple trees in an orchard where a fertilizer
test has been under way for the past five years. The
orchard has been in sod for many years. The trees, set
35 feet apart, have been lightly pruned; kept well sprayed,
and retained in a very productive state. Differences
as to vigor between the plots are obvious at once when the
orchard is viewed during the growing season. The plot
treatments together with the yields from the plots sampled

for enzyme determinations are shown in Table 12. In sampling,

- 31 -

discs were taken between 10 and 11 A. M. from the leaves
to the outside of 3 trees of the record row of the plot,
and from the same side at the same approximate vertical
level of the trees of each plot. Only leaves of equal
size and apparently equal vigor on non-bearing spurs of
the same length (l to 2 inches) were used. Necessary
equipment was taken to the orchard so that upon collecting
a sample it was immediately weighed, macerated and fully
prepared for'keeping and being made use of in the laboratory.
The method of determining the enzyme content was the same
as described on page 6.

The yields shown in Table 18 make it evident that
the several plots varied significantly in productivity and
hence in vigor as measured by yield. The enzymatic
activity of the samples from plots 2 and 3 composed of the
more vigorous and productive trees was 62% less in each
case than in plot 1.

Deductively, the conclusion that decreased
activity of oxidase and catalase is associated with in-
creased growth and size in plants, which has been reached
in this work and other work where disease etc. has been a
factor in checking growth or rendering it abnormal par-
ticularly the works of Bunzel (6) (7), Woods (24) and
Rose (19) would seem to be pertinent and correct. These
two enzymes are effective in oxidation processes within

the cells. Through processes of this nature compounds

 

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. 33 -

already synthesized and either assimilated or prepared

for assimilation are broken down to simpler substances.

even to carbon dioxide and water, and the energy they

contained released for the activities of the organism.

In other words, oxidase and catalase are primary in the

catabolic phase of metabolism. Since growth or increase

in mass is the resultant of the products of anabolism

being in excess of the amounts of elaborated materialsw

destroyed through catabolism, it must follow that it can

take place to a greater degree only as the activity of

those agencies concerned in catabolic processes is diminish-

ed relatively. Hence if it be true that growth-promoting

substances (nutrients) are effective through the enzymes

oxidase and catalase it is not surprising to find that they

bring about a reduction in the activity of the two catalysts.
The fact that catalase activity in seeds is higher

during germination as has been demonstrated by Shull (20)

and others does not conflict with the results and con-

clusions reached herein. It appears of course that this

increased activity of the enzyme is concomitant with the

beginning and acceleration of growth of the seed embryo,

but the situation is not closely analogous to that which

sbtains in the green leaf or stem. Synthesis of foods

from raw materials is not taking place in the seed and it

is not eXposed ordinarily to light and the other energiz-

ing factors of aerial environment. Yet the need for

- 34 -

energy is high and the supply of it must come from the
decomposition of stored materials through oxidation by
means of these oxidizing enzymes. Perhaps other enzymes,
concerned in hydrolytic reactions, are more positively
correlated with the nourishment and actual increase of the
embryo, and here as in the green leaf the increased activity
of oxidase and catalase in meeting the requirement of
energy tend at the same time to reduce the amount of assimila-
ble growth-promoting material. Where the stored supply of
food is large relative to the requirement of the embryo
prior to the appearance and functioning of the green leaves
as in.most seeds, the diminution of this supply occasioned
through oxidations affected by oxidizing enzymes would not
be critical nor very apparent as a check on growth. Hows
ever it is very noteworthy that experimental data available
on the rate of increase in growth from the time of the
beginning of seed germination show that there intervenes

a period at the start during which there is either no in-
crease in mass or even an actual reduction. Growth curves
with increase in size plotted against time show this as a
common characteristic. For example the publication of
Briggs, Kidd and West (5) wherein they have analyzed and
presented graphically the data compiled by Kreuster et.al.
respecting the growth rate of maize from the time of sow-
ing the seed. 0n the generalized form of the growth rate
curve (page 121) there is an initial portion covering the

‘45

livvlnlk/
at: 2
.

-35-

first three week period I'before the assimilatory organs

are able to counter balance the loss in dry weight due to
respiration, and the rate of growth is consequently
negative or nil". Quoting further from page 114, the
authors say: "In following the curves in Fig. 3 from the
date of sowing there is seen to be an initial phase last-
ing for about three weeks during which the rate of growth
is negative, or in other words the plant is actually losing
in weight. This phase of negative growth persists until

a point in the development of the plant is reached at

which approximately four leaves have appeared. During the
time occupied by germination, before the appearance of
these leaves, the negative rate of growth is clearly to be
attributed to a loss of carbohydrate through respiration.
The order of magnitude of the loss in dry weight through
respiration in germinating seeds is 3 to 6 percent of their
dry-weight per day at 16°C (Garreau (5))! Of equal interest
is the fact that the generalized form of the growth-rate
curve (page 121) shows two minima which precede two sub-
sidiary maxima, these maxima.coinciding "with the time of
the record of the appearance of the male and female flowers
respectively". The comment of the authors on this is as
follows, theparenthesis having been inserted; "The
minima x, y(indicating a decrement in growth) which

precede these maxima, correSpond with the earliest stages
of flower develoPment, and are possibly due to increased

_respiration (which predicates increased oxidase and cat-

- 55 -

alase activity) during that period.”

As additional evidence in support of the ex-
istence of a reverse relation between growth and the
activity of respiratory enzymes the work of Priestley and
Evershed (17) may be mentioned. They studied the root
growth of Tradescantia and Lycepersicum, based on increase
in the dry and wet weights of the roots from cuttings. The
grand period curves which were obtained proved to be series
of S-shaped curves. The subsidiary maxima of the curves
corresponded with the times of appearance of successive
crops of roots subordinate in the order of branching.

These maxima were preceded by minima which corresponded it

would seem to the periods of the origin and differentiation
of these branches in the pericycle region, and consequently
were coincident to the times of accelerated respiration in

that same region.

Lest it be supposed that the rise in the growth»
rate curves following the depressions discussed above,
particularly that which marks the early stages of germina-
tion, be due not to the actual decrease of the activity
of the enzymes under consideration and hence of respi-
ration intensity but wholly to the initiation and rapid
increase in the assimilatory functions of the leaves
relative to an unchanged respiratory rate attention should
be called to the fact that there are considerable data
which show that the intensity of respiration declines with

-57-

increased age of the plant. .A single citation, that of
the work of Kidd, West and Briggs (15) on Helianthgs
‘gggggg both in the laboratory and in the field, will be
sufficient. Using the respiratory index, which they define
as the respiration, measured by the rate of carbon dioxide
emitted, per gram of dry weight at 10°C when the amount
of respirable material is not limiting and where the ex-
ternal concentration of oxygen is that of the atmosphere,
they conclude from numerous experiments that the respiratory
index of the entire plant declines continuously as age in-
creases. A typical specimen of their results is the in-
stance where entire plants at 2 days after germination
gave 5 mgm. of carbon dioxide per gram of dry weight per
hour, while plants 136 days old produced only 0.59 mgm.
of carbon dioxide per gram of dry weight per hour. The
stem, leaves and flowers separately considered showed
decreases with age similar to that of the entire plant.
The fact that growth requires the energy re-
leased in the oxidations accomplished by the enzymatic
action is the thing which underlies any supposition that
growth and the activity of oxidase and catalase are
positively correlated. It should be remembered however.
that the processes of growth are catabolic as well as ana-
bolic and too, are not the only ones taking place in the
plant which use up energy, and furthermore that there is

no evidence to support any theory which would embody the

-38-

concept of there being an exact quantitative balance be-
tween the necessary amount of energy, and the activity of
energy producing agencies. This would attribute to plant
organisms a degree of economy and an inherent capacity for
conservation of energy which they mayor may not and most
likely do not possess.

Investigators have endeavored for some time to
determine whether catalase or oxidase is the more closely
connected with respiration. Perfect agreement on the
question has not been reached, the chief difficulty seem-
ing to have been the fact that the two enzymes are so
closely related that their separation for purposes of
experimentation is beyond our methods at present. What
evidence there is favors the conclusion that catalase
bears a closer relationship to respiration than oxidase
does. Reed's (18) data showed that the two enzymes are
independent of each other and that in the ripening of
fruit catalase increased while oxidase stayed constant, or
practically so. Crocker and Harrington (9) found catalase
activity ran parallel to respiration in the fruits of
Andropgggghalepgnges,but did not do this in seeds of
Amaranthug. Appleman (2) determined the comparative
activity of these enzymes in the expressed Juice of the
potato tuber and found a close positive correlation be-
tween catalase content and respiration intensity but no

such relation as regards oxidase. The data presented in

. 59 -

Table 11 confirm the results of these investigators though
the reasoning employed is necessarily somewhat indirect,
since direct determinations of respiratory intensity were
not made. The coefficient of correlation between average
size of plant and oxidase activity was -.246i,075, while
5031.085.
with catalase it was -.95fig.999. The difference is very
great and also wholly significant. The negative character
of these coefficients may indicate that as the activity of
the enzymes was lowered by the nutrient treatments of the
soil, growth was accelerated. Remembering that these
particular enzymes seem to be the principal ones effective
in respiration it means that as their activity was reduced
the intensity of respiration declined as well, this decrease
being of such.magnitudes that growth was increased be-
cause of a rise in the ratio of’anabolism to catabolismt
Hence it follows that since the coefficient of correlation
for catalase activity and growth was=near¥yifo§rrtimes-as
great as that for oxidase and growth, catalase appears to
stand in a closer relationship to respiration than oxidase.
In other words, a greater depression of catalase than of

oxidase was antecedent theoretically at least to lowered

respiration and consequently to increased growth and size.

3mm!

In these investigations the effect of certain
nutrient conditions of the soil on the activity of oxidase
and catalase in lettuce, radish and spinach plants and the

relation of this effect to growth have been studied. The

results were as follows:

1. is between size of plant and oxidase activity
correlation was slightly but significantly negative in

character.

2. Correlation between.size of plant and cat-
alase activity was strongly negative,thstngzszmast*per=
-feetiyfisst

5. In so far as the soil treatments affected
growth and size of plant they were antecedent to decreas-
ed oxidase and catalase activity when accelerating growth

and increased activity when growth was retarded.

ACKNOWLEDGMENTS

The writer wishes to make acknowledgment to
Dr. John W. Grist for many criticisms and suggestions
in planning and conducting the work outlined in this ex-
periment, and for assistance in the preparation of the
manuscript; and to Professor V. R. Gardner for reviewing

the manuscript.

l.

2.

3.

4.

5.

6.

'7.

8.

9.

- 42 -

LITERATURE CITED

Appleman, G. 0. Relation of catalase and oxidase to
respiration in plants. LMd. Agri. Exp. Sta. Bul. 191.
1915.

Appleman, C. 0. Relation of oxidases and catalase to
respiration in plants. Amer. qur. Bot. 5:225. 1916.
Appleman, G. 0. Respiration and catalase activity in
sweet corn. Am. Jour. Bot. 5:207. 1918.

Auchter, E. C. Is there normally a cross transfer of
foods, water and mineral nutrients in woody plants?
Md. Agri.-Exp. Sta. Bu1.257. 1925.

Briggs, G. E., Kidd, P. and West, C. A quantitative
analysis of plant growth. AmulAppl. Biol. 7:105. 1920.
Bunzel, H. H. (A biochemical study of curly-top of
sugar beets. U. S. D. A. Bur. of Plant Ind. Bul. 277.
1915.

Bunzel, H. H. Oxidases in healthy and curly-dwarf
potatoes. Jour. Agri. Res. 2:575. 1914.

Cook, M. T., and Taubenhaus, J. J. The relation of
parasitic fungi to the contents of the cells of the

host plants. Del. Agri. Exp. Sta. Bul. 97. 1912.
Cracker, Wm. and Harrington, G. T. Catalase and

oxidase content of seeds in relation to their dormancy,
age, vitality and respiration. Jour..Agri. Res.
15:137e 19180

10.

11.

12.

13.

14.

15.

16.

- 43 -1

Harvey, R. B. Relation of catalase, oxidase and El‘
concentration to the formation of over-growths. Amer.
qur. Bot. 7:211. 1920.

Harvey, R. B. An apparatus for measuring oxidase and
catalase activity. Jour. Gen. Physiology 2:255. 1920.
Heinicke, A. J. Factors influencing catalase activity
in apple-leaf tissue. Corn. Univ. Agri. Exp. Sta. Msm.
62. 1925. ‘

Heinicke,.A. J. Catalase activity in dormant apple
twigs: its relation to the condition of the tissue,
respiration and other factors. Corn. Univ. Agri.

Exp. Sta. Hem. 74. 1924.

Jones, C. H. Method for determining the lime re-
quirement of soils. Jour. Assoc. of Official Agri.
Chemists 1:45. 1915.

Kidd, P., West, C. and Briggs, G. B. A.quantative
analysis of the growth of Helianthus annus. Part 1.
The respiration of the plant and of its parts through-
out the life cycle. Proc. Roy. Soc., London, B,
92:568. 1921.

Moore, B. and Whitley, E. The properties and class-
ification of the oxidizing enzymes, and analogies be-
tween enzymic activity and the effects of immune bodies

and complements. Biochemical Jour. 4:156. 1909.

.
n
O r
. P
. I v
. . 1
u
r . u . C
v A
o .
.
4 I
O O
s
.
a
e e e
D 1
.
A v
. .
1 e I
.
I ’
.
. .
.
. n
. Y
e
. .
.
~
e .
.
. i .
O
a. .
. _

 

17.

18.

19.

20.

21.

22.

23.

24.

-44..

Priestley, J. H. and Evershed, A. F. C. H. A quanti-
tative study of the growth of roots. Ann. Bot. 56:225.

.1922.

Reed, G. B. The relation between oxidase and cata-

lase in plant tissues. Bot. Gaz. 62:409. 1916.

Rose, D. H. Oxidation in healthy and diseased apple
bark. Bot. Gaz. 60:55. 1915.

Shull, C. A. and Davis, W. B. Delayed germination and
catalase activity in Ianthium. Bot. Gas. 75:268. 1925.
Sullivan,.M..X. The action of salts used as fertilizers
on plant enzymes. Jour. Biol Chem. 6:XLIV. 1909.

True, R. H. and Stockberger, W. W. Physiological
observations on alkaloids, latex and oxidases in Papaver
somniferum. Amer. Jour. Bot. 5:1. 1916.

Weiss, F. and Harvey, R. B. Catalase, hydrogen-ion
concentration and growth in the potato wart disease.
qur..Agri. Res. 21:589. 1921.

Woods, A. F. Observations on the mosaic disease of

tobacco. U. S. D. A. Bur. of Plant Ind. Bul. 18. 1902.

  

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