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Bulletin 374

September, 1935

CHEMICAL INVESTIGATIONS OF THE
TOBACCO PLANT .

V. CHEMICAL CHANGES THAT OCCUR
DURING GROWTH

HUBERT BRADFORD VICKERY, GEORGE W. PUCHER,
CHARLES S. LEAVENWORTH and ALFRED J. WAKEMAN

With the technical assistance of Laurence S. Nolan

Agrirultural lExpnim^nt fttatum
32eto ^aben

CONNECTICUT AGRICULTURAL EXPERIMENT STATION

BOARD OF CONTROL

His Excellency, Governor Wilbur L. Cross, ex-officio, President

Elijah Rogers, Vice-President Southington

William L. Slate, Treasurer New Haven

Edward C. Schneider, Secretary Middletown

Joseph W. Alsop Avon

Charles G. Morris Newtown

Albert B. Plant Branford

Olcott F. King South Windsor

Administration.

STAFF

William L. Slate, B.Sc, Director.

Miss L. M. Brautlecht, Bookkeeper and Librarian.

Miss Katherine M. Palmer, B.Litt., Editor.

G. E. Graham, In Charge of Buildings and Grounds.

Analytical
Chemistry.

E. M. Bailey, Ph.D., Chemist in Charge.

C. E. Shepard 1

Owen L. Nolan

Harry J. Fisher, Ph.D. \ Assistant Chemists.

W. T. Mathis

David C. Walden, B.S. J

V. L. Churchill, Sampling Agent.

Mrs. A. B. Vosburgh, Secretary.

Biochemistry.

H. B. Vickery, Ph.D., Biochemist in Charge.

Lafayette B. Mendel, Ph.D., Research Associate (Yale University).

George W. Pucher, Ph.D., Assistant Biochemist.

Botany.

G. P. Clinton, Sc.D., Botanist in Charge.

E. M. Stoddard, B.S., Pomologist.

Miss Florence A. McCormick, Ph.D., Pathologist.

A. A. Dunlap, Ph.D., Assistant Mycologist.

A. D. McDonnell, General Assistant.

Mrs. W. W. Kelsey, Secretary.

Entomology.

Forestry.

Plant Breeding.

Soils.

Tobacco Substation
at Windsor.

Assistant Entomologists.

W. E. Britton, Ph.D., D.Sc, Entomologist in Charge, State Entomologist.

B. H. Walden, B.Agr.

M. P. Zappe, B.S.

Philip Garman, Ph.D.

Roger B. Friend, Ph.D.

Neely Turner, M.A. j

John T. Ashworth, Deputy in Charge of Gipsy Moth Control.

R. C. Botsford, Deputy in Charge of Mosquito Elimination.

J. P. Johnson, B.S., Deputy in Charge of Japanese Beetle Quarantine.

Miss Helen A. Hulse } _ .

Miss Betty Scoville f Secretaries.

Walter O. Filley, Forester in Charge.

H. W. Hicock, M.F., Assistant Forester.

J. E. Riley, Jr., M.F., In Charge of Blister Rust Control.

Miss Pauline A. Merchant, Secretary.

Donald F. Jones, Sc.D., Geneticist in Charge.
W. Ralph Singleton, Sc.D., Assistant Geneticist.
Lawrence C. Curtis, B.S., Assistant.

M. F. Morgan, Ph.D., Agronomist in Charge.
H. G. M. Jacobson, M.S., Assistant Agronomist.
Herbert A. Lunt, Ph.D., Assistant in Forest Soils.
Dwight B. Downs, General Assistant.
Miss Geraldine Everett, Secretary.

Paul J. Anderson, Ph.D., Pathologist in Charge.

T. R. Swanback, M.S., Agronomist.

O. E. Street, Ph.D., Plant Physiologist.

Miss Dorothy Lenard, Secretary.

Printing by Quinnipiack Press, Inc., New Haven, Conn.

CONTENTS

PAGE

Introduction 557

Description of Collections 561

Preparation of Material 563

Analytical Methods 565

Analysis of dry leaf, stem, and inflorescence samples 565

Analysis of leaf and stem extracts 569

Analysis of sediments, sludges, and extracted residues 570

Expression of the data 571

The Growth of the Tobacco Plant 573

Growth in terms of fresh weight 573

Growth in terms of dry weight 574

Organic solids and ash 574

Organic acids 577

Carbohydrates 580

Ether extractives 582

Nitrogenous constituents 583

Nitrate nitrogen . '. 585

Nicotine nitrogen 586

Ammonia and amide nitrogen 586

6N acid hydrolyzable ammonia 593

Amino nitrogen 593

Relative Distribution of the Constituents of the Tobacco Plant 595

The Work of Smirnov 602

Summary 604

Bibliography 607

Appendix 608

CHEMICAL INVESTIGATIONS OF THE TOBACCO PLANT
V. CHEMICAL CHANGES THAT OCCUR DURING GROWTH

Hubert Bradford Vickery, George W. Pucher,
Charles S. Leavenworth and Alfred J. Wakeman*

INTRODUCTION

The study from the chemical point of view of the growth of plants is
seriously circumscribed by the lack of suitable and accurate methods of
analysis. Although the importance of an understanding of the reactions
that occur in the cells of a developing plant can hardly be overestimated,
the progress that has been made is most disappointing. It is obvious
that a knowledge of the qualitative composition of plant tissues must pre-
cede the investigation of the quantitative relationships between the con-
stituents; but this knowledge is still so elementary that one must be sat-
isfied, for the most part, with quantitative measurements of groups of
constituents rather than with individuals, with "forms" of nitrogen rather
than with definite compounds, and with such thoroughly indefinite fac-
tors as the water-soluble organic solids, the ether extractives, or the
fermentable carbohydrates.

Nevertheless, one has only to turn to the early literature of the subject
to appreciate the very real advances that have come since Emmerling made
his first attempt in 1880 to solve the problem of the origin of the amino
acids found in plant tissues. Emmerling's investigation (11, 12, 13) f
represents 20 years spent upon the analysis of a series of bean plants
(Vicia faba, major) collected in the season of 1880. The time required
gives some notion of the labor expended on these analyses. The nitrogen
determinations were carried out by the Varrentrap and Will method of
combustion with soda-lime, the amino nitrogen determinations by an
elaborate but cumbersome modification of the Sacchse-Korman method —
the forerunner of the present day Van Slyke method. Few determina-
tions depended on volumetric methods ; even ammonia was estimated
from the weight of the ammonium chloride produced by allowing a sample
of the tissue intimately mixed with lime to stand for three days in an
evacuated apparatus that contained an absorption column moistened with

Note: The chemical investigations of tobacco herein described were carried out as part of a
general project under the title "Cell Chemistry," by the Department of Biochemistry of the
Connecticut Agricultural Experiment Station, New Haven, Conn. The Department has enjoyed
the benefit of close cooperation from the Tobacco Substation. The expenses were shared by the
Connecticut Agricultural Experiment Station and the Carnegie Institution of Washington.

*With the technical assistance of Laurence S. Nolan.

tNumbers refer to bibliography, page 607.

558 Connecticut Experiment Station Bulletin 374

dilute hydrochloric acid. Time was a minor consideration in those davs,
and Emmerling was a conservative with respect to the adoption of newer
and more convenient analytical methods. Nevertheless his results are of
great significance. He expressed his data in terms of percentage of the
dry substance and also in grams in 1000 plants. The curves that illus-
trate the accumulation of dry substance — nitrogen, amino nitrogen, and
protein nitrogen in leaves, stems, pods, seeds, and roots — furnish a vivid
picture of the growth of the plant, and of the migration of organic solids
and of nitrogenous substances from the leaves to the seeds as these matured.
The interesting part played by the seed pods, as an intermediate store-
house of nitrogenous substances later furnished to the seeds, is particu-
larly well shown.

Emmerling's conclusions in the broader field of general metabolism are
still of importance. He pointed out that the reserve protein of the sprout-
ing seed undergoes enzymatic hydrolysis to its components ; these migrate,
together with nitrogen-free reserve substances (sugar, fat), to the centers
of new growth where, under the influence of respiration with its attendant
liberation of energy, the nitrogenous products are resynthesized into cell
proteins. In the absence or deficiency of nitrogen-free reserve substances,
the respiration is maintained at the expense of the nitrogenous components
and, as a result, under these circumstances asparagine or glutamine be-
come prominent products of the reactions. These substances in turn may
provide a source of the ammonia required for protein synthesis. After
the leaves have developed, assimilation of carbon becomes possible, and
the incoming nitrogenous substances are rapidly converted to tissue protein
inasmuch as energy derived from respiration is readily available. The
further development of the plant involves the transport of inorganic nitrog-
enous substances from the soil, and their transformation into a form
(probably ammonia) suitable for assimilation by combination with the
carbon compounds produced by the process of oxidation in the leaves.
these products then serving for the synthesis of cell protein.

Emmerling's investigations of plant growth are unique for their time.
His views on the general subject of nitrogen metabolism are closely re-
lated to those of Schulze (32) who had reached similar conclusions from
his extensive studies of the growth of seedlings. Most investigators of
this period were chiefly concerned with the rate of accumulation of inor-
ganic nutrients in the plant, the interest being mainly confined to the
study of the drain upon the soil incident to the growth of the crop, and
to the interpretation of the results in terms of the correct practice for the
maintenance of fertility. The value of such studies to practical agriculture
is obvious and, inasmuch as the chemical methods of ash analysis have
long been highly developed, accurate and significant data were readily
to be obtained.

Although data referring to the total nitrogen, the organic dry matter,
the crude fiber, the ether extract, the carbohydrates, and the crude protein
were frequently included in the reports on the study of the inorganic
constituents, these factors were arrived at by purely conventional routine
methods, and the data seldom admit of interpretation in terms of chemical-
ly distinct individual organic substances. Even when data on the nitrate,

Introduction 559

the ammonia, and the amide nitrogen are given, critical examination of
the methods of analysis that were employed frequently gives rise to doubt
that the results have much significance in revealing the details of the
physiology of organic growth.

The most important investigations of the inorganic constituents of
plants throughout a part or the whole of the growth period have been
contributed by experiment station workers in this country and abroad.
Schweitzer (33), in 1889 at the Missouri Station, studied the growth of
maize by analysis of the plant at approximately weekly intervals from the
seed to maturity. He dissected the plant into root, stem, and leaf, and
later into husk, tassels, silk, and ear as well, dried the material in an oven
to constant weight, and determined the composition of the ash of each
part with great care. The data were expressed for the most part in
terms of per cent of dry weight, but the quantities in grams per plant were
likewise given. Jones and Huston (19) in 1914 published the report
of a somewhat similar investigation of the maize plant, the data for which
had been collected in 1903 at the Indiana Station. In addition to the
fresh and dry weights, and the detailed analyses of the ash of the different
parts of the plant, they also determined the nitrogen, the crude fiber, the
starch, the "fat", or ether extract, and the so-called "albuminoid" and
"amide" nitrogen. The data were expressed in terms of pounds per
10,000 plants, or approximately one acre.

Perhaps the most elaborate of these early studies of plant growth is
that of Wilfarth, Romer and Wimmer (51) at Bernburg, Germany, in
1903, who described investigations with wheat, barley, potatoes, peas, and
mustard, giving analyses of the parts of the plants for dry matter, starch,
potassium, sodium, nitrogen, and phosphorus. The data were expressed
as per cent of the dry matter and in pounds per acre.

White (50), working at the Georgia Experiment Station, in 1914
recorded the ash composition of the cotton plant at four stages in its
growth in terms of grams per plant and per cent of dry matter.

In recent years Knowles and his collaborators in England have again
taken up this type of investigation. They have studied wheat (20) and
also the sugar beet (21). Their analyses include data for the ash, and
ash constituents, the dry matter, the nitrogen, and, in the case of the
sugar beet, for the carbohydrates as well. The data are expressed both
as per cent of dry matter, and as grams in a definite number of plants.
They likewise provide calculations of the distribution of the more im-
portant constituents in the different parts of the plant.

The literature of plant composition at different seasons, as affected
by various methods of culture or of fertilization of the soil, is, of course,
very extensive. Attention need be drawn only to the work of Shive and
his students (34, 38, 39) and to the investigations of the apple tree by
Thomas (41) as examples. A full discussion of the early work of this
nature on fruit trees is given by Gardner, Bradford and Hooker (15)
and by Chandler (6).

The behavior of the nitrogen, dry matter, and of the ash constituents
of the leaves of woody plants throughout the growing season has been
studied by a number of workers. Tucker and Tollens (42) described a

560 Connecticut Experiment Station Bulletin 374

very thorough and careful investigation of the leaves of Platanus occi-
dentalism in which data were expressed in terms of leaf area, in grams
per definite number of leaves, and in per cent of the dry matter. Schulze
and Schiitz (31) studied the composition of maple leaves collected in the
morning and evening at monthly intervals in the growing period, basing
their results on the absolute quantity in 200 leaves of equal size. Swart
(40) gave data at two stages (early summer and late autumn) for a
number of species, and included in his report a very complete review of
the earlier investigations on the problem of autumnal migration of nitro-
gen and inorganic constituents out of the leaves.

More closely allied in point of view with our own work is the investi-
gation of Chibnall (7) who studied the composition of runner bean
(Phaseolus vulgaris v. multiftoris) leaves throughout the growing season,
with special attention to the protein and to certain of the simpler nitrog-
enous constituents. He demonstrated that the leaf protein, although
varying quite widely in the relative proportion present at different times,
remained practically unchanged in composition. Culpepper and Caldwell
(9) have recently studied the composition of the rhubarb plant (leaves,
petioles, and root) at various stages of growth. They were particularly
interested in the titratable acidity, the organic solids, both soluble and
insoluble, and in the nitrogen.

Very little work of the general character of most of the papers men-
tioned appears to have been done on the tobacco plant. Davidson (10)
in 1895 published analyses of Virginia tobacco plants for moisture, ash,
and ash constituents at several stages of growth from seedling to maturity,
and Carpenter (5) in 1893, in the course of a study of the composition
of cured tobacco, gathered a few data on plants grown in North Carolina.
No further detailed study of the growth of the tobacco plant has come to
our attention until the work of Smirnov and his collaborators at Krasno-
dar appeared in 1928 (35, 36).

Smirnov's technic differed fundamentally from that of the American
investigators. His interest was centered upon the relationship between
the composition of the leaf and the surface area, and the changes in this
relationship with the growth of the plant. The area of each leaf was
determined from the weight of a section cut with a die of known area, the
midrib being removed before weighing the fresh leaf. The cut sections,
after being weighed, were employed for the determination of the dry
weight; the residual material was preserved by treatment with hot alcohol,
and was stored for analysis under alcohol. Determinations were made
of the water-holding capacity of the dry tissue at 95 per cent humidity,
of the peroxidase activity, of the carbohydrates (starch, dextrin, saccha-
rose, maltose, monosaccharides), and organic acids (Fleischer's method
(14) ; see also Vickery and Pucher (44, p. 163) ) and of the total nitro-
gen, protein nitrogen (Barnstein's method (3) ), formol titratable nitro-
gen (Sorensen (37) ), ammonia, nicotine, and nitrate nitrogen. The
samples examined ranged in age from seedling to mature plants the leaves
of which were beginning to turn yellow ; both normal and topped plants
were employed. The detailed discussion of Smirnov's results will be
deferred until after our own data have been presented (see p. 602).

Description of Collections 561

The present study of the growth of the tobacco plant arose from the
need of data on the composition, with respect to the carbohydrates, organic
acids, and nitrogenous substances, at different stages of development of
the plant, in order that material might be more intelligently selected for
studies of the metabolism of this plant. We have attempted to collect
chemical data from which as complete a picture as possible can be drawn
of the synthesis of organic substances in the whole leaf and stem tissue,
and later in the pods as well, during growth from the seedling stage to
senescence. Many of the methods of analysis employed have been de-
veloped in this laboratory for special application to the tobacco plant.

The plants employed were of the variety known as Cuban Shade, or
perhaps more generally, as Connecticut shade-grown tobacco. This variety
is characterized by a high content of nitrogen in terms of the dry weight
of the leaf, and a low content of carbohydrate, and is grown commercially
exclusively for the production of cigar wrappers (16). The agricultural
conditions under which the plants employed for the experiment were pro-
duced are thoroughly standardized and were carefully controlled by mem-
bers of the scientific staff of the Tobacco Substation at Windsor, Connec-
ticut. We are deeply indebted to them for their cooperation. The plants
were grown under shade tents on a soil each acre of which had been
dressed with a mixed fertilizer composed of :

2700 lbs. ground tobacco stems

1000 '

' cottonseed meal

100 '

; potassium nitrate

200 '

' fish meal

200 '

' "Calurea"

100 '

; precipitated bone

100 '

' magnesian lime

The composition of this mixture was such as to supply approximately
200 lbs. of nitrogen, 100 lbs. of phosphoric oxide, 200 lbs. of potassium
oxide and 75 lbs. of magnesium oxide per acre.

DESCRIPTION OF COLLECTIONS

A record of the dates at which collections were made, together with
the weather conditions, is given in Table 1.

Collection A consisted of seedlings uprooted from the seedbed the day
after the field had been set with plants taken from the same bed. The
stems were separated from the roots just above the cotyledons. The
leaves that were to be extracted were rinsed from adhering sand and
dirt in a large vessel of water. The water was subsequently filtered, and
the dry weight of the soil collected was subtracted from the weight of
the leaves. The leaves that were to be dried were placed in a ventilated
oven. After being dried they were carefully agitated to detach any sand,
and this was collected and weighed. The corrected weights of the two
stem samples were similarly obtained.

Collection B was taken after the seedlings had been set for 19 days. It
is to be noted that the leaves of the plants in this sample were newly

562 Connecticut Experiment Station • Bulletin 374

developed ; the leaves of the seedlings are almost invariably sloughed off
after transplantation. The plants varied in size, most being within the
range of 5 to 7 grams; a few were considerably heavier, the largest weigh-
ing 20 grams. Many plants had been attacked by the potato flea beetle,
and a few leaves had been so badly damaged that it was necessary to dis-
card them. Most of the plants had four leaves. The preparation of the
samples and the corrections for adhering soil were made as before.

Collection C at 26 days was made by cutting the stem at the ground
level. The plants bore four to eight leaves with a number of smaller ones
at the top of the stem ; this top portion was added entire to the leaf
fraction. Corrections for adhering soil were made as before.

Table 1

Days from Date

Collection setting 1933 Weather

A 1 June 1 Overcast.

B 19 June 19 Clear and cool; rain previous day.

C 26 June 26 Overcast ; cold nights but clear most of week.

D 35 July 5 Irrigated with equivalent of 1.7 inches of rain

previous day. Overcast and cold for two

days. Growth normal for season.
E 40 July 10 Cool and mostly fine; plants a little backward.

F 47 July 17 Rainfall during past two months about half

normal and plants about one week retarded.

Light rain during night before collection.
G 54 July 24 No rain for past week ; hot and humid.

H 61 July 31 Very hot; no rain since July 16.

I 75 Aug. 14 Rain during previous night, the first since July

16. Weather overcast.
J 97 Sept. 5 Heavy rain during previous week but fine on

date of gathering.
K 110 Sept. 18 Heavy rain Sept. 13 to 17 (2.5 inches).

Collection D at 35 days consisted of plants that bore eight to twelve large,
and a number of small leaves. There was very little soil on these plants
and this was confined to the lower leaves. The stems had become notably
hard and woody.

Collection E at 40 days consisted of rapidly maturing plants. Bottom
suckers had been broken off and discarded a few days before the collection.
The correction for soil on the leaves was negligible from this point on.
Owing, to the increased size of the plants, only 16 were taken for Sample
I, and 14 for Sample II.

Collection F at 47 days consisted of plants about 4 feet high. The dry
season had held the plants back about one week of their normal rate
of development. Only 10 plants each for Samples I and II were collected
from this point on.

Collection G was made at 54 days, three days after the commencement
of the first picking (lowest four leaves) from all the plants in the field save
those reserved for this experiment. The weather was hot and humid and
the plants were somewhat wilted. A few plants in the field had begun
to blossom.

Collection H at 61 days was the first in which an inflorescence sample
was taken ; this consisted mostly of buds. The weather had been extreme-

Preparation of Material 563

ly hot for some days and there had been no rain. The second picking of
the crop had been completed during the previous week.

Collection I at 75 days consisted of mature plants with well-developed
flowers. There had been rain the night before the collection. The gather-
ing of the crop (fourth picking) had been completed.

Collection J, made 97 days from setting, was taken from plants left in
the field under a small shade tent for this experiment. A heavy storm
a few days earlier had damaged some of the plants severely. Erect and
unharmed plants were selected. The seed pods were well developed, but
a few blossoms remained on some of the plants.

Collection K at 110 days, the last of the series, was made chiefly to
provide data on the influence of the setting of seed. Over 2.5 inches of
rain had fallen during the previous five days, and most of the plants were
badly damaged in spite of the protection afforded by the shade tent. The
best possible selection of plants was made under the circumstances. Many
bore fully ripe seed, although a few were still in flower.

In making the collections care was taken to select plants of a uniform
state of development as near to that of the average condition of the plants
throughout the field as possible. All collections were made at about
9 A. M., the material being then transported to the laboratory at New
Haven.

PREPARATION OF MATERIAL

Each collection was subdivided into two" roughly equal lots designated
Samples I and II. The plants of each sample were dissected into leaf
and stem, or later into leaf, stem, and inflorescence or pod portions.1

The leaves and stems of Sample I from each collection were separately
extracted three times each, successively, with boiling water acidified to
pH 3 to 4 with sulfuric acid, according to the technic described in Station
Bulletin 324 (45). The complete leaf extracts amounted to from 25 to
40 liters, the stem extracts to from 15 to 30 liters. Previous to extrac-
tion, the stems were cut into short lengths and split into several pieces.

Each extract was allowed to stand overnight, and was then decanted
from a small quantity of sludge. This sludge was separated by elutriation
from any sand that had also settled, and was then washed by centrifuga-
tion and dried. When necessary, the sand was collected and weighed,
the weight being applied as a correction on the fresh weight of the sample.
The extracts were filtered through paper pulp and rapidly concentrated
in vacuo in stills equipped with vapor coolers (46) until the sediment
which separated made further concentration difficult. The sediments were
then centrifuged, washed free from nicotine, dried, weighed, and ground
for analysis. The solutions were made to a definite volume and preserved
with a liberal quantity of toluene. The residues of both leaf and stem
tissue were shredded and dried in a ventilated oven, being subsequently
weighed and ground to a powder for analysis.

1 A more elaborate dissection of the plants into lower, middle, and upper leaf and stem por-
tions could not be attempted in the present investigation in spite of the great desirability of data
of this sort. The term "stem" in the present publication refers to the main stalk of the plant;
to the tobacco technologist, however, the term refers onlv to the midrib of the leaf.

564 Connecticut Experiment Station Bulletin 374

Sample II from each collection, after dissection into leaf and stem
portions, was dried at a temperature of from 80 to 90° and then weighed
and ground to a fine powder. The oven employed for the stems was
ventilated by a rapid stream of air heated to approximately 100°. Drying
was complete within a few hours.

The inflorescence or pod samples consisted of flowers, and more or
less well-developed seed pods. Each pod was cut from its pedicel sepa-
rately, the denuded stalk being added to the stem sample. In all save one
case, the inflorescence samples were dried for analysis. This was found
to be necessary as the presence of the oil in the maturing seeds rendered
hot water extraction impracticable.

The material accumulated for analysis consisted of the following prep-
arations for each of the eleven collections of plants.

Leaf extract Stem extract

Leaf sludge Stem sludge

Leaf sediment Stem sediment

Extracted leaf residue Extracted stem residue

Dried leaf Dried stem

In addition, dried inflorescence specimens from both Sample I and
Sample II were obtained from the last four collections.

In order to simplify the presentation, it has seemed desirable to combine
certain items of the analytical data. The sludges that separated from the
leaf and stem extracts obviously consisted to a considerable extent of
coagulated protein ; this material should therefore be regarded as a part
of the insoluble extracted residue, and the data for ash, inorganic solids
and nitrogen of the sludges have been combined with the corresponding
figures from the analyses of these residues. In the case of the younger
plants, the solids and nitrogen of the sludge derived from the leaf tissue
formed a very considerable part of the whole. The sludge from the stem
extracts was so small, however, that complete analyses could be obtained
only for the last three collections.

The sediments that separated from the voluminous leaf and stem ex-
tracts, after these had been concentrated to a relatively small volume,
obviously represent soluble constituents of the respective tissues. The
data for ash, organic solids, and nitrogen have therefore been added to
the corresponding data from the analyses of the extracts. The sediments
consisted very largely of calcium sulfate, and it was clear that the sulfate
radical was derived from the sulfuric acid added to maintain a sufficient
acidity during the extraction. The weights of these ashes obviously do
not accurately represent the inorganic constituents of the tissue. The
assumption was therefore made that the calcium was derived from calcium
salts of organic acids and, further, that no significant error would be
committed by regarding the whole of the ash from the sediments as calcium
sulfate. The quantity of calcium oxide equivalent to this ash weight was
therefore calculated, and the result was added to the inorganic solids of
the respective extracts. We have previously shown (48) that, when this
artifice is employed, the ash determined on dry leaf tissue corresponds
closely with the sum of the ashes of the extract and extracted residue.

Analytical Methods 565

On standing, the concentrated leaf and stem extracts slowly deposited
a further sediment. This was small in amount, and was therefore removed
by centrifuging before aliquots were withdrawn from the extracts for
analysis; the error involved in neglecting this additional sediment is
undoubtedly insignificant.

ANALYTICAL METHODS
Analysis of Dry Leaf, Stem, and Inflorescence Samples

The moisture content of the thoroughly mixed specimen was obtained
by drying weighed portions in porcelain capsules to constant weight in a
vacuum desiccator over sulfuric acid. This usually required four to five
days.

The ash was obtained by igniting the dried residue in an electric muffle.

Total nitrogen was determined by the Kjeldahl method, with mercury
as catalyst, after previous reduction of the nitrate with dilute sulfuric acid
and reduced iron powder, according to the method of Pucher, Leaven-
worth and Vickery (25).

Ammonia nitrogen was determined by distillation in the presence of
magnesium oxide in vacuo. The apparatus consisted of a 500 cc. Pyrex
round-bottomed flask fitted with two necks. A bulb trap was attached
to the central and larger neck by a standard taper ground-glass joint, and
the vapor tube leading from the trap was bent to descend vertically into
the receiver. This consisted of a 25 by 250 mm. heavy-wall test tube
graduated at 50 cc, and fitted with a two-hole rubber stopper to accom-
modate the vapor tube and the wide-bore tube1 leading to the vacuum
pump. The receiver was immersed in a beaker of cold water in lieu of a
condenser. ^The second and smaller neck of the distillation flask was fitted,
also by means of a standard taper ground-glass joint, with an air inlet
tube bent so as to reach nearly to the bottom of the flask. The air entering
through this tube was washed with dilute acid contained in a small gas-
washing bottle. ^

To conduct a determination, a 0.500 gm. sample of the dry powdered
tissue was transferred to the distillation flask, and 5 cc. of a 12.5 per cent
suspension of light magnesium oxide powder in water were added, together
with 30 cc. of water. The receiver was charged with 3 cc. of 0.1 N
hydrochloric acid. The apparatus was closed and evacuated, care being
taken to allow sufficient air to enter through the air inlet tube to agitate
the contents of the flask thoroughly. The air supply was then cut down
to a slow current, and a water bath, previously heated to 40°, was raised
under the distillation flask so as to cover it almost completely. Distillation
was allowed to continue for 15 minutes, when the vacuum was released
and the receiver was disconnected. The vapor tube was washed into
the receiver with a little water, and 5 cc. of Nessler's solution (22) were

1 Loss of a little ammonia from the distillate may occur unless the tube leading to the pump
is of sufficiently wide bore, or is furnished with a small bulb. This tube should ascend vertically
for 6 to 10 cm. above the stopper before being bent. It should invariably be washed back into
the receiver at the termination of a distillation.

566 Connecticut Experiment Station Bulletin 374

added to the contents with agitation ; the distillate was then diluted to
50 cc. The color produced was read in a Pulfrich photometer with light
filter S-43, using an ammonia-free solution that contained 5 cc. of Nessler's
reagent diluted to 50 cc. in the blank cell. The ammonia value was read
from a calibration chart in which the extinction coefficient was plotted
against milligrams of ammonia nitrogen. Blank determinations on the ap-
paratus and reagents were made periodically, and were remarkably constant
at 0.006 to 0.008 mg. of ammonia nitrogen. It was found necessary to con-
duct a blank distillation at the beginning of each day's work and discard
the distillate. This served to remove a substance1 which accumulated in
the standing apparatus overnight and gave rise to a slight turbidity with
Nessler's reagent.

Quantities of from 0.02 to 2.0 mg. of ammonia can be determined with
an error not exceeding 0.005 mg. by this technic, provided photometer
cells are available that give readings between 30 and 80 per cent trans-
mission on the instrument. Usually the 5 or 10 mm. cells were used, but
a 30 mm. cell was required to read the blank determinations.

Check analyses demonstrated that nicotine is not volatilized under the
described conditions in sufficient quantity to interfere with the determina-
tion of the ammonia.2

Nitrate nitrogen was determined by the method of Pucher, Vickery
and Wakeman (27).

Glutamine amide nitrogen3 was determined by hydrolyzing 0.200 gm.
of the tissue in the presence of 10 cc. of a phosphate-borate buffer at pH
7.0 for 2 hours in a boiling water bath. The hydrolysis was carried
out in a test tube closed with a stopper that carried a short length of fine-
bore tubing to serve as a condenser. The reaction at the end of this
operation was in the range pH 6.2 to 6.6. The resulting suspension was
transferred with 10 to 15 cc. of water to the ammonia distillation apparatus
already described; 2.0 cc. of 1 N sodium hydroxide, and 15 cc. of water
were added. The ammonia was then distilled off, and the increase over
the quantity of ammonia already present in the tissue was taken as the
glutamine amide nitrogen. Owing to the presence of the phosphate buffer,
the use of sodium hydroxide was found to be essential to successful quanti-
tative distillation of the ammonia. The presence of magnesium was found
to be objectionable because of the possibility that magnesium ammonium
phosphate might be formed in sufficient amount to prevent quantitative
removal of the ammonia within the time allowed for the distillation.

Asparagine amide nitrogen : An extract of the dry tissue was prepared
by suspending 2.50 gm. of the powder in 25 cc. of water in a beaker, and
boiling for 3 minutes with constant stirring. The solution was cooled and
the aqueous volume made to 50 cc. The suspension was then centrifuged,

1 This interfering substance was eventually found to arise from a spontaneous change in the
rubber stopper used to close the receiver. The difficulty could be avoided either by boiling this
stopper with alkali at the beginning of a day's work, or by simply running a blank distillation.
The latter seemed preferable.

- An improved modification of this method to determine ammonia is given by Pucher, Vickery
and Leavenworth. Ind. and Eng. Chem., Anal. Ed., 7: 152. 1935.

3 The data upon which this method to determine glutamine is founded are given by Vickery,
Pucher, Clark, Chibnall and Westall (in press). This paper also contains later improvements in
technic.

Analytical Methods 567

and aliquots of the clear fluid were withdrawn for determinations of aspara-
gine amide nitrogen, and of the amino and peptide nitrogen. Extracts
of the pod tissue were prepared in the same manner from the residues
after previous extraction of the substances soluble in ether.

Asparagine amide nitrogen was determined by heating 5 cc. of the
extract together with 1 cc. of 6 N sulfuric acid in a 25 by 250 mm. test
tube in a boiling water bath for 3 hours. The contents of the tube were
transferred to the ammonia distillation apparatus, 15 to 20 cc. of water,
5 cc. of N sodium hydroxide, and 5 cc. of 12.5 per cent suspension of
light magnesium oxide were added, and the determination of ammonia
was conducted as already described. The asparagine amide nitrogen was
calculated by subtracting the sum of the free ammonia nitrogen and of
the glutamine amide nitrogen from the ammonia nitrogen found after
hydrolysis of the extract with normal acid.

Amino nitrogen was determined by transferring 10 cc. of the extract
to the ammonia distillation apparatus where it was freed from ammonia
by distillation with magnesium oxide. The residue was treated with
2 cc. of glacial acetic acid to dissolve the magnesia, and was transferred
to a 50 cc. flask and made to volume. Amino nitrogen was determined
in 5 cc. aliquots of this solution in the Van Slyke manometric apparatus
according to the directions of Peters and Van Slyke (24). It should be
noted in passing that this method is much superior both in accuracy and
convenience to the earlier methods of Van Slyke that involve the special
amino nitrogen apparatus.

Peptide nitrogen was determined by transferring 5 cc. of the extract
to a 25 by 250 mm. test tube together with 5 cc. of 12 N sulfuric acid ;
the mixture was heated in a boiling water bath for 6 hours, and was then
washed into the ammonia distillation apparatus with 10 to 15 cc. of
water; 10 cc. of 5 N sodium hydroxide, and 5 cc. of 12.5 per cent mag-
nesium oxide suspension were added, and the ammonia was distilled off
and determined in the usual way. The residue was acidified with 2 cc.
of glacial acetic acid and was made, to 50 cc. Amino nitrogen was de-
termined as before in this solution. The peptide nitrogen represents the
increase in amino nitrogen on hydrolysis with 6 N acid.

The presence of glutamine in the tissues of the tobacco plant renders
the results of this method somewhat untrustworthy. Chibnall and Westall
(8) have shown that the deamidation of glutamine in hot neutral solution
is accompanied by a loss of amino nitrogen. This probably results from
ring formation with the production of pyrrolidone carboxylic acid. If,
during the preparation of the tissue for analysis (e.g. during drying), a
part of the glutamine present has undergone deamidation, this part will be
represented in the material subjected to analysis by pyrrolidone carboxylic
acid, a soluble substance which, on hydrolysis with strong acid, yields
glutamic acid with a resultant increase in the amino nitrogen of the
solution. That this source of error may be significant is shown in the
discussion of the analysis of the stem samples on a later page.

Total organic acidity and oxalic acid were determined as described
by Pucher, Vickery and Wakeman (28). According to this method, the

568 Connecticut Experiment Station Bulletin 374

organic acids are extracted from the acidified tissue with ether and trans-
ferred to aqueous alkaline solution. The total organic acidity is obtained
by titration at the quinhydrone electrode between the limits pH 7.8 and
2.6. Oxalic acid can be titrated only to the extent of 50 per cent under
these conditions; the oxalic acid is therefore separately determined in a
portion of the organic acid solution, by precipitation as calcium salt, and
a correction of the titration value is made. A correction factor is also
applied to allow for the fact that malic and citric acids are titrated only
to the extent of approximately 90 per cent under the described conditions.

Malic acid was determined by the method of Pucher, Vickery and
Wakeman (29). According to this method an aliquot part of the organic
acid solution, obtained as described above, is oxidized with potassium
permanganate in the presence of bromine; the volatile oxidation product
is distilled with steam and converted to its dinitrophenylhydrazone, which
is filtered off and dissolved in pyridine. A portion of the pyridine solution
is made alkaline with sodium hydroxide, and the blue color produced is
read in a Pulfrich photometer. The quantity of malic acid is then obtained
from a calibration curve in which the extinction coefficient is plotted
against known quantities of malic acid that have been treated in the same
manner. The method is capable of estimating from 0.1 to 2.5 mg. of
malic acid with an accuracy of ± 5 per cent.

Citric acid was determined by the method of Pucher, Vickery and
Leavenworth (26) which is a modification of the pentabromoacetone
method of Hartmann and Hillig (18). A portion of the organic acid
solution, obtained as described above, is oxidized with potassium per-
manganate in the presence of potassium bromide, the pentabromoacetone
produced is extracted with petroleum ether, dehalogenated with sodium
sulfide, and the bromide ion is titrated with silver nitrate. The method
is capable of estimating from 1 to 20 mg. of citric acid with an accuracy
of ± 5 per cent.

The ether extract was determined by drying the sample in an atmos-
phere of hydrogen for 5 hours at 100° and subsequently extracting with
absolute ether for 15 to 20 hours. The residue, after evaporation of the
ether, was dried to constant weight in a steam oven.

For the determination of the carbohydrates, an alcohol extract of the
dried tissue was prepared. The alcohol was purified before use by dis-
tilling absolute alcohol over potassium hydroxide, and the neutral distillate
was diluted to 75 per cent by volume with water. Five grams of the
dried tissue were placed in a paper thimble and extracted with the diluted
alcohol in a continuous extraction apparatus for at least 6 hours. The
extract was concentrated in vacuo to remove alcohol, and the aqueous
residue was made to 100 cc. and preserved with toluene.

Total soluble carbohydrate was determined in 10 cc. aliquots as
described by Vickery, Pucher, Wakeman and Leavenworth (48, on pp.
74 and 75). Minor difficulties encountered in the determination of the
unfermentable carbohydrate in the solutions prepared with mercuric sul-
fate led to the development of a modified method of preparing the solutions
for this determination.

Analytical Methods 569

A 10 cc. aliquot of the solution obtained by alcohol extraction of the
tissue was treated with 1 cc. of 5 N sulfuric acid and heated at 75° for
12 minutes to invert the sucrose. The solution was then diluted with
30 to 40 cc. of water, and neutralized to litmus with cold saturated barium
hydroxide solution; 10 cc. of 0.2 N oxalic acid were added, and the
solution was made to 100 cc, centrifuged, and then poured through a
dry filter to remove any turbidity present. A 50 cc. aliquot of the filtrate
was gently shaken with 5 gm. of Lloyd reagent in a 125 cc. Erlenmeyer
flask for 4 minutes ; 3 gm. of permutit were added, and the flask was
again shaken gently for 3 minutes. The solution was then centrifuged
clear. A blank of 10 cc. of distilled water was carried through the same
operations. The total reduction was determined on 2 cc. portions of the
Lloyd reagent filtrate by heating with 2 cc. of 0.01 N ferricyanide and
6 cc. of water for 15 minutes in a boiling water bath and proceeding as
described by Vickery, Pucher, Wakeman and Leavenworth (48, p. 75).

The unfermentable carbohydrate was determined after removal of
the fermentable sugar with yeast. A yeast suspension was prepared as
described by Vickery, Pucher, Wakeman and Leavenworth (48, p. 76)
and 4 cc. were transferred to a centrifuge tube and centrifuged at high
speed until the cells were firmly packed together. The fluid was poured
off and the residual moisture was removed from the walls of the tube
and the surface of the yeast with dry filter paper ; 10 cc. of the Lloyd
reagent filtrate were added, and the cells were thoroughly mixed with
the solution and allowed to react for 10 minutes at room temperature.
A blank determination on 10 cc. of the Lloyd reagent blank solution was
similarly conducted. After the fermentation was complete, the yeast
was centrifuged down, and 2 cc. aliquots of the clear fluid were treated
with ferricyanide as before.

Comparisons of the sugar content of solutions as prepared in this way,
and of solutions prepared according to the mercuric sulfate clarification
technic, showed that equally satisfactory results were obtained by either
procedure.

Crude fiber was determined according to the standard conventional
procedure (2).

Analysis of Leaf and Stem Extracts

Samples of the extract were centrifuged until perfectly clear before
aliquot parts were withdrawn for analysis ; the small quantity of sludge
so removed was neglected.

Total solids were determined by evaporating 10 cc. samples in porce-
lain capsules to dryness on a steam bath. The residue was then dried
to constant weight in a vacuum desiccator over sulfuric acid. Constant
weight was attained in about 120 hours with the extracts from the younger
collections ; the older ones required from 15 to 20 days.

The ash was obtained by igniting the dry residue in an electric muffle
at 560 to 600°.

Total nitrogen was determined in 5 cc. aliquots by the method em-
ployed for the dry-leaf samples.

570 Connecticut Experiment Station Bulletin 374

Nicotine was determined in 5 cc. aliquots, after distillation with
steam from alkaline solution, by precipitation with silicotungstic acid (1).

Ammonia nitrogen was determined in 5 cc. aliquots of the extract as
already described.

Amide nitrogen was determined by hydrolysis with 1 N sulfuric
acid for 3 hours in a boiling water bath. To this end, 5 cc. of extract, 5 cc.
of water, and 2 cc. of 6 N sulfuric acid were heated as already described in
a 25 by 250 mm. test tube for 3 hours. The ammonia was then determined
in the usual way. The increase in ammonia brought about by the acid
hydrolysis was calculated as amide nitrogen.

The amide nitrogen of these extracts does not include the glutamine
amide nitrogen ; any glutamine present in the tissue would have been
hydrolyzed during the preparation of the extract.

6 N acid hydrolyzable ammonia : The ammonia liberated by hy-
drolysis for 6 hours with 6 N sulfuric acid was determined in order to
obtain evidence for the presence of substances that can be decomposed
only by severe hydrolysis with the production of ammonia. Certain of
the purines are partially decomposed in this way. The data are not to
be considered as determinations of adenine or other purine derivatives ;
at present we prefer to interpret this factor in a purely empirical manner.
To carry out the hydrolysis, 10 cc. of the extract were diluted to 50 cc,
and 3 cc. of this were mixed with 3 cc. of 12 N sulfuric acid in a test
tube, as already described, and heated in a boiling water bath for 6 hours.
The contents of the tube were transferred to the ammonia distillation
apparatus, 8 cc. of 5 N sodium hydroxide, 10 cc. of 12.5 per cent mag-
nesium oxide and 10 to 15 cc. of water were added, and the ammonia was
distilled in vacuo and estimated in the usual way. The increase over the
ammonia produced by 1 N acid hydrolysis was calculated and is given
as the 6 N acid hydrolyzable ammonia.

The so-called easily hydrolyzed amide nitrogen was calculated by
subtracting the free ammonia nitrogen of the dry leaf and stem samples
(Samples II) from the free ammonia nitrogen of the respective hot water
extracts (Samples I).

Analysis of Sediments, Sludges, and Extracted Residues

Total nitrogen was determined by the Kjeldahl method, moisture
and ash by the methods described under analysis of the dry leaf and
stem samples.

No attempts were made in the present investigation to obtain a direct
measure of the protein nitrogen of the leaves or stems. Although several
methods which are supposed to furnish information regarding the pro-
tein nitrogen of plant tissue are described in the literature, none of these,
in our opinion, provides trustworthy data. For the present, therefore,
we have been compelled to assume that the nitrogen which remains in-
soluble, when a sample of fresh tissue is thoroughly extracted with hot
water, is at least a representation of the quantity of protein nitrogen of
that tissue. Such results are undoubtedly too high, and it is the purpose
of investigations, now being conducted in this laboratory, to arrive at an

Analytical Methods 571

idea of how much too high they may be. Information already at hand
suggests that appreciably more than 10 per cent of the insoluble nitrogen
belongs to non-protein substances.

For reasons that have been discussed elsewhere (43), we have also
omitted all reference to the basic nitrogen of the extracts from the tissues.

Expression of the Data

The problem of presenting data derived from the analysis of a series
of living organisms of different size and age in such a way as to provide
a vivid and easily grasped picture of the nature of the changes that have
occurred during growth is extremely difficult. The usual bases upon
which such analytical data are calculated in terms of per cent all vary
during the period of growth, and the only factor that remains constant is
the biological unit itself, in this case the individual plant. Calculation of
the ratio between, for example, the fresh weight and the total nitrogen
shows how this ratio progressively changes during the growth period,
but gives no idea whatever of the actual quantities either of nitrogen, or
of total plant mass, involved. It refers exclusively to relative concen-
tration of the nitrogen in the plant, and therefore cannot serve as a meas-
ure of the growth process. The same restriction applies to calculations
on any other of the customary bases of percentage, such as dry weight,
residual dry weight, i.e. the dry weight exclusive of the carbohydrates
(23), or the total nitrogen. If, however, the biological unit is accepted as
a basis of comparison, that is, if the comparisons are made on a basis of
quantity of any constituent per individual plant, it is possible to present
a picture of the growth process that clearly reveals the quantities involved.
Furthermore data so calculated readily lend themselves to recalculation on
any percentage basis that may subsequently seem desirable.

The fundamental data of the present investigation have therefore been
computed in terms of grams per plant, and the curves plotted from these
data show the actual change in the weight of each factor, within the limits
of experimental error, as growth progressed. The most serious disad-
vantage of this method is the inconveniently small magnitude of the
quantities involved in the analysis of the earlier collections ; this makes
graphical presentation on a uniform weight scale difficult. On the other
hand, the analyses of these earlier collections, being based upon a much
larger number of individual plants, are undoubtedly relatively more ac-
curate than those of the later collections where only a few plants could
be dealt with.

Reference should be made at this point to the able presentation given
by Gouwentak (17) of the difficulties of the proper expression of the data
of plant analysis. Her critique of the methods that have been commonly
employed merits the most careful attention. For comparisons between
the results of analysis of small numbers of leaves at short time intervals,
she prefers to express the data in terms of leaf surface area, and rejects
the methods of expression in terms of percentage of the fresh or dry
weight. In spite of certain advantages, however, this method is clearly
impractical for use in our case.

572 Connecticut Experiment Station Bulletin 374

In addition to expressing the data in terms of weight per plant, in some
cases the distribution of certain of the constituents among leaf, stem,
and pods has been calculated. These results are presented in a separate
section together with calculations of the percentage distribution of the
various forms of nitrogen of the leaves and stems. In a few cases, how-
ever, it has seemed desirable to give calculations of the percentage dis-
tribution of certain constituents, notably the organic acids and carbohy-
drates, along with the discussion of the weight data.

The data have been plotted in the charts upon a uniform time scale ;
the scale of ordinates, however, varies from figure to figure, being selected
in each case so as to give curves that are as clearly separated from each
other as possible. In most cases the points of observation are represented
by circles, and the progression of the quantity is represented by a solid line.
Occasionally it has been necessary to employ crosses and dotted lines to
facilitate the exposition, or to prevent possible confusion of the curves.

The analytical data are collected into tables that appear at the back of
the present publication. The second column shows the figure in which
each set of data is plotted ; the third column indicates to which of the two
samples from each collection the data in the following columns refer.

It will be noted that the greater part of the data is derived from the
analysis of the dried leaf and stem samples herein designated as Sample
II. In certain cases the same constituent was determined both in Sample
II and in Sample I which had been subjected to hot water extraction.
When this was done the agreement between the results from the early
collections was, for the most part, satisfactory, but the agreement between
the later collections of only 10 plants per sample was occasionally not
good. Although great pains were taken at each of these later collections
to obtain 20 plants of uniform size and development for the two samples,
it is clearly impossible to rely implicitly on so small a group as 10 plants
for such detailed analyses as the present study involves. Furthermore, as
the plants aged, individual variations became more pronounced, and the
accidents incident to the bad weather of the last few weeks of the season
inevitably had their effect upon the results. The assumption involved in
such a method of collection is that each lot of 10 plants represents with
sufficient accuracy what the next previous lot of 10 plants would have
been had they remained in the field. Our experience indicates that this
requirement is extremely difficult to satisfy.

In this connection the experience of Knowles, Watkin and Hendry
(21) is of interest. They based their data upon collections of 64 plants
(sugar beets) but, in order to obtain samples of manageable size, were
compelled to divide the individual plants of the older collections into
quarters, retaining only one-quarter of each. In spite of the great care
with which their material was handled, minor irregularities of the same
type as those we have encountered appeared in their data.

Growth of the Tobacco Plant
THE GROWTH OF THE TOBACCO PLANT

573

Growth in Terms of Fresh Weight

The fresh weight of one plant, calculated from the average weight of
all the plants taken at each collection, is plotted in Figure 1. During the
first three weeks after being set, there was very little increase in weight
of the tops ; the original seedling leaves seldom survive the operation of
transplantation, and practically the whole of the weight at 19 days from
setting represents the development of the seedling bud. Doubtless con-
siderable growth of the root system occurred in this interval inasmuch
as the tops more than trebled their weight in the ensuing week, after the
plant had become established. Thereafter the rate of growth rapidly ac-
celerated, the slope of the growth curve reaching a maximum between the
40th and 47th days ; subsequently the rate diminished until the plant began
to produce seed, when no further increase in fresh weight occurred.

Fig. 1

1200

240 r

0 Days 40

0 Days 40

120

The relative proportions of stem and leaf tissue changed materially
during the period of rapid growth. At 35 days only about one-quarter of
the total fresh weight consisted of stem tissue, but at 61 days the weight
of stem tissue equaled that of the leaves and subsequently exceeded it.
The weight of stem tissue reached a maximum at 75 days and thereafter
changed but little ; the weight of the leaf tissue diminished during the
development of the inflorescence. The precise magnitude of these later
changes is in some doubt because of the sampling error involved in the
selection of the older plants.

The water content of the plants of Sample II of each collection is plotted
in Figure 2. The curves are less smooth than those in Figure 1 because

574 Connecticut Experiment Station Bulletin 374

the data are calculated from the analysis of a smaller number of individual
plants ; they show the rapid rise to a maximum water content at the 75th
day, and a subsequent loss of water from the leaves as the inflorescence
developed. The water content of the stems somewhat exceeded that of
the- leaves after the 75th day.

Growth in Terms of Dry Weight

The dry weight per plant calculated from the analyses of Sample II
from each collection is shown in Figure 3. After an initial period (40
days) of slowly accelerating rate of growth, the plant began to lay down
dry matter at a practically constant rate ; the growth curve from 40 to
75 days can be quite satisfactorily smoothed to a straight line that does
not depart from the observations by a quantity greater than the experi-
mental error. The two last observations show that the rate of accu-
mulation of dry matter subsequently slowed down somewhat. A careful
examination of the growth curve indicates .that the maximum rate of
growth occurred in the period from, roughly, 40 to 54 days. It is im-
portant to note the contrast between the rate of growth as measured by
fresh weight and that now under discussion. The maximal growth rate
in terms of fresh weight was attained between the 40th and 47th days,
and this rate subsequently diminished rapidly and ceased at the time of
pod development; the maximal rate in terms of dry weight, although
attained at approximately the same time, continued somewhat longer and
did not diminish rapidly; growth continued to the end. The character
of the growth of the plant is quite different when regarded from these two
points of view.

Figure 3 also shows the rate of accumulation of dry matter in the leaves,
stems, and inflorescence. The quantity of dry matter in the leaf tissue
reached a maximum at about the 75th day, the rate of growth from 26
days to that point being nearly linear. Later, as the inflorescence began
to develop, the leaves lost a little in dry weight. The stem tissue, on the
other hand, continued to increase in weight up to the final point of obser-
vation ; the period of maximal rate of increase corresponds with that of
the whole plant and, as in that case, the diminution in rate was not marked.
The inflorescence increased in dry weight at a fairly constant rate and
sufficiently rapidly so that, at the end of the period of observation, the
weight of the pods amounted to nearly one-fifth of the entire dry weight
of the plant. It should be noted that the seeds in a large proportion of the
pods taken at the last collection were far from fully developed, and it is
clear, from the tendencies of the curves, that seed development places a
heavy drain upon the organic substance of the leaves.

Organic Solids and Ash

The quantities of organic solids, plotted in Figure 4, give curves that
closely resemble those of the total dry weight and require little additional
comment. The most notable feature is the evidence of continuing ac-
cumulation of organic solids at a relatively steady rate long after the total
mass of the plant as measured by the fresh weight (see Figure 1) had
ceased to increase. The change is due to the development of the in-

Growth of the Tobacco Plant

575

florescence and of the stem, these over-balancing the loss of solids by the
leaves.

Figure 5 shows the quantities of water-soluble organic solids in the
leaves and stems of the plants of Sample I from each collection. A careful

Fig. 4

50

40

30

20

10

0 Days 40

06

150

Fig. 5

90

30

ne-.A

0 Days 40

o£

0 Days 40

120

Fig. 7

0*

0 Days 40

12

06-

Fig. 8

0 Days 40

120

Fig. 9

0o

0 Days 40

120

consideration of the data of the last three collections shows that sampling
errors render difficult strict comparisons between the plants of Sample I
used for water extraction, and those of Sample II which were dried.
The earlier collections, however, yielded data that mutually support each
other and are consistent. If the curves of Figure 4 are compared with

576 Connecticut Experiment Station Bulletin 374

those of Figure 5, it is clear that the distribution of water-soluble organic
solids between leaves and stem followed a course quite different from the
distribution of total organic solids. At all save one point (26 days) the
leaves contained more soluble organic solids than the stem. Further-
more, the rate of accumulation in the leaves during the first 40 days
was relatively rapid whereas, in the same period, the soluble solids in
the stems scarcely increased at all. Subsequently soluble solids increased
in both leaves and stem at about the same rate up to the 61st day; there-
after the leaves continued to increase, but the stems dropped behind.
The marked deflection of the leaf curve at the last collection is probably
due to translocation to the seeds during the previous interval.

The rate of accumulation of the insoluble organic solids is shown in
Figure 6. The curve for the leaves lies above that of the stem during the
first 54 days. The curve for the stem, however, inflects upwards sharply
at 40 days, and shows an exceptionally rapid rate of deposition of in-
soluble organic solids throughout the period of most rapid growth of
the plant, doubtless due to the formation of woody tissue in this period.
The quantity of insoluble solids in the stem exceeds that in the leaves
from the 54th day on.

The weights of the ash obtained on incinerating specimens derived
from Sample II of each collection are plotted in Figure 7. Aside from
the irregularities due to somewhat high values for the data from the 54-
day collection, and somewhat low values for those from the 61 -day col-
lection, the curves indicate an accumulation of inorganic substances at a
rapidly accelerating rate during the first 40 days, followed by a period
in which ash was absorbed from the soil at a relatively constant rate
until the plants reached their maximum size as indicated by the fresh
weight (75 days). The data suggest that no significant further ab-
sorption of inorganic matter took place.

The ash in the leaves is at all times materially higher than that in the
stem in spite of what appears to have been a migration of ash constituents
from the leaves to the pods as the seeds developed. The ash in the stems
reached a maximum on the 75th day and maintained this unchanged until
the termination of the experiment notwithstanding that the stems were
rapidly accumulating organic substances during the last five weeks. The
ash of the inflorescence also failed to increase materially after the 75th
day although organic solids were being rapidly laid down.

The distribution of ash constituents between the water-soluble and in-
soluble fractions obtained from the plants of Sample I of each collection
is shown in Figures 8 and 9. The soluble ash of the leaves exceeded that
of the stems at all save one point (26 days) and showed in general a
behavior similar to that of the soluble organic solids. The soluble ash of
the stem, after a period of lag up to the 40th day, increased at a rate
only slightly less than that of the soluble ash of the leaves. There is no
evidence of a marked drop in soluble ash in the stem at the last collection,
and the general behavior of the curves suggests a migration of ash con-
stituents from leaves to inflorescence at this time. The data of Figure
7, derived from the plants of Sample II, support the same view.

Growth of the Tobacco Plant 577

The insoluble ash constituents of the leaves also exceeded those of the
stem at all times. The curve for the leaves shows the same drop at the
time of the final collection as does the curve for soluble ash, again sug-
gesting migration from leaves to inflorescence of inorganic constituents.

Organic Acids

The total organic acidity of the tobacco plant, plotted in Figure 10,
represents corrected titration values between pH 7.8 and 2.6 of the or-
ganic acids that can be extracted by ether from the acidified tissue. The
data are expressed in milliequivalents per plant instead of in grams, as
this permits closer comparison of the chemical effects of the different
acids ; furthermore it is possible to assign an accurate magnitude to the
unknown acids when so expressed inasmuch as the factor for the con-
version of these unknown acids from equivalents to grams is, of course,
not known.

The acidity of the whole plant follows a curve that is closely similar to
the curve which represents the ash content of the plant. This relationship
is not one of chance ; unpublished data accumulated in this laboratory on
a wide variety of samples of cured tobacco show a close correlation be-
tween the ash content and the total acidity. The curve of the acidity of
the leaves is also in almost every detail like that of the ash of the leaves ;
the acidity of the stems and the ash of the stems differ only with respect
to the final observation. It is to be noted that, particularly with the
younger plants, by far the greater part of the total acidity is to be found
in the leaf tissue. The acidity of the stem increased, however, at a fairly
constant rate, and in the older plants makes up approximately one-third
of the total. The development of the fruit was accompanied by a decrease
in the acidity of the leaves of an order of magnitude comparable to the
acidity found in the seed pods. Here also attention should be directed to
the closely similar behavior of the ash ; the picture suggested is one of
migration of acids in salt combination from leaves to seed pods.

In Figure 11 are given the detailed data on the composition of the
leaves with respect to the individual organic acids. The chief acid of
tobacco leaf tissue is malic acid; in this particular lot of leaves oxalic
acid was next in quantitative importance, citric acid being present in
somewhat smaller amounts. The total quantity of unknown acids lies
between the quantities of citric and oxalic acids. Little is known of the
chemical nature of the portion recorded as unknown acid; it represents
the difference between the total acidity and that due to the sum of the
three known acids. Qualitative studies of tobacco leaf tissue have shown
the presence of small amounts of succinic, and possibly of fumaric acid,
together with appreciable proportions of acids that form esters both of
low and of high boiling points (44). In addition there is a certain
quantity of substances the behavior of which suggests the presence of
phenolic groups.

A brief study of the acids of the leaves of collection F at 47 days
has shown that approximately one-third of the acidity belonging to un-
known acids is insoluble in water although soluble in ether. All that
can be said, therefore, about the chemical nature of the substances in the

578

Connecticut Experiment Station

Bulletin 374

unknown fraction is that the evidence points to the presence in it of a
complex mixture of acidic substances.

The rapid increase of malic acid to a maximum value at 75 days was
followed by a moderate drop during the period of fruiting. The oxalic
acid behaved in a similar manner; the citric acid, however, reached a

Fig. 12

250

Days 40

0 Days 40

0 Days 40

maximal value somewhat earlier and, with minor fluctuations, maintained
it to the end. The marked drop at 61 days in all of the curves is un-
doubtedly due to the somewhat low relative weight of the plants of Sample
II collected at that time, and may be regarded as sampling error; an

Growth of the Tobacco Plant 579

analogous drop was not evident in the data obtained from the leaves of
Sample I of the same collection.

The distribution of the acids in the pods (Figure 13) is quite different
from that of the leaves. The unknown acids make up the greater part
of the acidity ; malic acid is present in considerable amount, but oxalic
and citric acids are found only in minor quantities.

In order to give an idea of the relative acid distribution of the leaves,
the data are plotted in another manner in Figure 14. The quantities
shown in solid lines represent the percentage of the total acidity as each
of the three known acids. The sum of the malic and citric acid is shown
by the lower broken line, and the sum of the malic, citric and oxalic acids
by the upper broken line. This line, therefore, is a plot of the relative
proportion of unknown acid if the ordinates are reversed and read down-
wards. The area between the malic acid curve and the lower broken line
represents the relative proportion of citric acid ; that between the two
broken lines, the relative proportion of oxalic acid ; and the area above
the upper broken line represents the relative proportion of unknown acids.

The variations shown by the curves in the relative proportions of the
acids in the young plants are undoubtedly significant. These collections
included much larger numbers of plants than did the later collections, so
that the sampling error is much smaller. The chemical data are of equal
accuracy throughout.

In the youngest plants the proportion of unknown acids (36 per cent)
approached the proportion of malic acid (47 per cent ) ; the proportions
of oxalic and citric acid were relatively small. During the earliest phases
of growth the oxalic acid increased rapidly in relative quantity and the
unknown acids decreased ; malic acid decreased slightly, and citric acid
increased a little. Then, at the point when the seedlings had become
established and the period of rapid growth was beginning, citric acid
for a short time increased and both malic and oxalic acids decreased.
From then on, the relative proportion of oxalic acid stayed constant,
citric acid diminished within two weeks to its previous level and there-
after remained constant, and malic acid, at first rapidly and then slowly
increased; at 61 days, however, it also reached a level that was main-
tained to the end of the period studied. If attention is confined to the
period after 40 days, which is that in which the most rapid growth of the
plant as a whole took place, the relative proportions of the three most
important organic acids are practically constant. In other words these
three acids are produced in the metabolism of the leaves, during the period
of most rapid growth, in amounts that are essentially constant relative to
each other. This implies that the unknown acids are likewise produced at
a fixed rate. Reference to Figure 10 shows that from 26 to 75 days the
total acidity of the leaves increased at a rate that can be satisfactorily
expressed by a straight line, but then suddenly began to diminish. Figure
14 shows that, in spite of the sudden cessation of deposition of acid in
the leaves, or perhaps the evidence of migration of acid out of the leaves,
the relative proportions of the three acids still present remained the
same. If organic acids actually were transported out of the leaves in the
period after 75 days, it is necessary to suppose that the individual acids

580 Connecticut Experiment Station Bulletin 374

were removed in the same relative proportion as that in which they were
present at this time. An alternative to this view is to suppose that the
three acids were transformed into non-acidic substances at the same
relative rate. Clearly there is a close and intimate interrelationship in the
metabolism of these acids in leaf tissue. It is difficult, in the face of these
observations, to regard oxalic acid merely as an end-product of carbo-
hydrate, or of organic acid oxidation, which is stored in the leaves as the
presumably inert calcium oxalate.

The organic acids of the stems are plotted individually in Figure 12.
The most striking feature of the diagram is the far more important role
played by the acids of unknown nature in the stem tissue than in the
leaves. The observations after 75 days suggest a degree of relative ir-
regularity that is not nearly so marked in the other data for the stems
of these later collections and therefore can hardly be ascribed exclusively
to sampling errors. It may be noted, however, that the total acidity of
the stems at 75 days (Figure 10) and also the ash content (Figure 7)
are both relatively high if the curves for both sets of observations are
smoothed.

The malic acid steadily increased in quantity throughout the period
of growth and showed no effect of the onset of flowering. The oxalic
acid increased to a maximum that was attained at 75 days, and subse-
quently did not change. Citric acid was present only in small proportion
throughout ; there is evidence of a slight increase in citric acid in the stems
of the last collection.

The relative proportions of the total acidity of the stems present as
malic, citric and oxalic acids are plotted in Figure 15. Here again the
importance of oxalic acid in the metabolism during the earliest stages of
growth is manifest. The proportion of oxalic acid increased and that of
malic acid correspondingly diminished during the first 26 days. During
the ensuing two weeks the relative proportions of these two acids were
reversed, malic again exceeding oxalic ; subsequently the malic acid was
maintained at approximately a constant level, save for an apparent marked
drop at 75 days. The oxalic acid steadily diminished from 35 days to
54 days, although by only a small proportion, but later remained con-
stant. Citric acid apparently plays only a minor part in the metabolism
of the stems ; there was none detectable in the stems of the seedlings ; a
small proportion was subsequently slowly formed and remained almost
unchanged throughout.

The large proportion of acids of unknown nature in the stem tissue is
striking. It is clear that, as growth progressed, these acids increased
slowly in relative proportion. The whole question of the acid compo-
sition of the stem tissue awaits a more detailed investigation. A brief
study of the acids of collection F (47 days) showed that about two-
thirds of the unknown acids are insoluble in water although soluble in
ether, but no information regarding their chemical nature has yet been
obtained.

Carbohydrates

The soluble carbohydrates of the leaves, calculated arbitrarily as glucose,
are shown in Figure 16. The total soluble carbohydrate began to in-

Growth of the Tobacco Plant 581

crease very rapidly as soon as the plants became established. The curve
of fermentable carbohydrate, which probably represents mainly glucose,
differs, however, inasmuch as it shows a sequence of changes in rate of
accumulation. During the interval from 40 to 61 days the leaves scarcely
increased their store of fermentable sugar at all, but then, as the rate of
growth diminished, sugar again accumulated.

The chemical nature of the unfermentable carbohydrate has not as yet
been established. The quantity formed is at all points less than that of
the fermentable sugar save in the leaves in the very earliest stages (see
solid line of percentage in Figure 18). This form of carbohydrate does
not show any marked fluctuation in rate of accumulation in the leaves
during the period of most rapid growth, and consequently the curve
showing the percentage of fermentable carbohydrate (Figure 18) drops
rapidly in this period — i.e. from 40 to 54 days.

The soluble carbohydrates of the stems are shown in Figure 17. The
quantities present in the stems of the very young plants were smaller
than those in the leaves but, after 47 days, the quantities materially ex-
ceeded those in the leaves. The total carbohydrate increased steadily
throughout the period of most rapid growth, a drop in the rate of ac-
cumulation being noted only after 61 days when the growth rate of the
entire plant had markedly diminished. The fermentable carbohydrate
follows the curve of total carbohydrate in every detail, the only apparent
check in the rate of accumulation being in the period from 75 to 97
days.

The behavior of the fermentable sugar suggests that transport into the
stem from the leaves occurred so rapidly, during the period of most
active growth, that continual accumulation was possible in spite of utiliza-
tion for the formation of new tissue. The leaves, as has already been
noted, were unable to do much more than hold their own with respect to
the quantity of fermentable sugar they contained in the period from 40
to 54 days, but the stems continually increased their store. Rapid growth
is obviously correlated with the presence of adequate supplies of fermenta-
ble carbohydrate in the stems of the plants, but can go on even when there
is little storage in the leaves in excess of that transported to the stems.

The unfermentable carbohydrate of the stems increased regularly, and
at a fairly constant rate throughout the period studied. There are no
significant inflections that correspond with either the period of rapid
growth, or of blossoming and fruit development. In view of our lack
of knowledge of the chemical nature of this factor, interpretation is dif-
ficult.

The crude fiber, determined by the conventional procedure, is shown
in Figure 19. As might be expected, the crude fiber of the stem mounted
rapidly throughout the period of most active growth ; it continued to in-
crease, however, even in the plants of the later collections at a rate that
was scarcely diminished.

The crude fiber of the leaves behaved in a totally different manner;
it- accumulated at a slow but steady rate for 54 days and thereafter re-
mained practically constant. Reference to Figure 4 shows that the be-
havior of the crude fiber is closely like that of the organic solids of the

582

Connecticut Experiment Station

Bulletin 374

leaves. The scale on which the crude fiber of the leaves is plotted is too
small to show the minor variations clearly, but, if the data are plotted
on a sufficiently large scale, a curve is obtained that faithfully follows the
irregularities of the curve for the leaves in Figure 4. It may be inferred
that the tobacco plant reaches a stage, at the end of approximately two
months, at which a fairly. definite amount of organic substances has been
laid down in the leaf tissue ; the subsequent growth of the plant as a whole
does not greatly alter the total quantity of organic matter in the leaves,
the phenomena then observed are those of interconversion of organic
substances rather than accumulation. The contrast with the behavior of

Fig. 17

00

" Fig. 18

Carbohydrates:

Per cent Fermentable

X ^*~« .x

75

1 fti *'* *•* y&\_

50

■ J

25
+^
c
a

u

) Days 40 80 120

0 Days 40

0 Days 40

the stem tissue is especially sharp ; continued accumulation of organic
solids takes place (see Figure 4), especially of the woody tissue upon
which the strength of the entire structure depends.

Ether Extractives

The quantities of ether-soluble substances extracted from the dried leaf
and pod tissue are plotted in Figure 20. This fraction includes chlorophyll
in addition to the hydrocarbons, waxes, sterols, and true fats that make
up the bulk of the material. The data show a progressive increase in
the ether-soluble substances of the leaves for 47 days, followed by a

Growth of the Tobacco Plant 583

period (47 to 75 days) in which the increase is almost linear. Subse-
quently there is a loss of ether-soluble substances from the leaves, but
whether or not this is connected with the development of the inflorescence
cannot be determined.

The ether extract of the pod samples reveals the rapid deposition of
true fat as the seeds ripen. No data on the composition of fully ripened
seed pods were obtained so that the maximum to which this curve may
rise is not certain. Our investigations of tobacco seed show (49), however,
that the dried seeds contain 42 per cent or more of their weight as ether-
soluble substance, most of which is an oil (30). An average plant may
be expected to produce approximately 44 grams of seed and, consequently,
the curve for the fat content of the ripened pods should ultimately reach
a magnitude of about 19 grams.1

Nitrogenous Constituents

The total nitrogen content of the plant, plotted in Figure 21, increased
with great rapidity from the earliest stage. The fresh weight of the
plants at 19 days was only from three to four times as great as that of the
seedlings, but the nitrogen content was nearly seven times as great. In
the interval from 19 to 26 days the nitrogen content increased fourfold
and in the next 9 days it increased nearly fivefold again. During the
period of most rapid growth, that is from 40 to 61 days, the plants steadily
assimilated nitrogen in the tops at the rate of 1 gram in about 8 days.2
The apparent loss of nitrogen from the tops in the last period studied is
a matter that requires further study.

By far the greater part of the nitrogen taken up by the plant in the early
stages of growth is assimilated in the leaves, and at all stages, save in
the aging plant, the quantity of nitrogen in the leaves is at least twice
as great as that in the stem. The onset of flowering places a relatively
sudden check upon the rate of storage of nitrogen in both leaves and
stem, and the tendencies of the curves suggest that nitrogen is more or
less rapidly translocated from other parts of the plant into the ovules as
these ripen.

The relative proportions of soluble and insoluble nitrogen in the leaves
and stem are shown in Figures 22 and 23. The insoluble nitrogen may,
for practical purposes, be regarded chiefly as protein nitrogen, and it is
clear that protein synthesis is extremely rapid even in the youngest plants.
The 19-day old leaves contained ten times as much insoluble nitrogen
as the seedling leaves, and the quantity increased more than fourfold
in each of the next two intervals of seven and nine days. During the
period of most rapid growth, insoluble nitrogen was laid down in the
leaves at the rate of 1 gram in 17 days. At the expiration of 61 days,
that is at the beginning of the period of flowering, deposition of insoluble

1 This admittedly very rough estimate is based on the following figures. An average plant
bears approximately 175 pods, each of which contains from 2,000 to 4,000 seeds — 3,000 may
be taken as an average. One plant therefore produces well over 500.000 seeds. The seeds run
from 11,500 to 13.000 to the gram, 12,000 being a rather accurate average. Much of the data
for this estimate is contained in Station Bulletin 326 (4) : other figures are based on unpublished
counts and weights of seeds made by Dr. Th. Berthold at the Tobacco Substation at Windsor, Conn.

aThis is at the rate of approximately 26.5 lbs. per acre in 8 days on the assumption of 12,000
plants to the acre.

584

Connecticut Experiment Station

Bulletin 374

nitrogen in the leaves abruptly ceased and, in the last interval studied,
insoluble nitrogen was apparently removed from the leaves.

The soluble nitrogen of the leaves, though less in amount than the
insoluble, followed a somewhat similar course during the first 47 days.
The rate of accumulation of soluble nitrogen then diminished somewhat,
but the curve clearly indicates an accumulation of soluble nitrogen through-
out the period in which the insoluble nitrogen of the leaves remained
constant. The marked drop in soluble nitrogen at the 110th day is
probably associated with the rapid development of the seeds.

3.0

1.5

o.o*

Fig. 22

Leaves:
Soluble and
Insoluble N

0 Days 40 80

120

0.0*

Fig. 23

Stems:
Soluble and
Insoluble N

0 Days 40

0 Days 40

0 Days 40

0 Days 40

The insoluble nitrogen of the stems (Figure 23) did not begin to
accumulate in significant amounts until the plants were 26 days old, and
the rate of deposition thereafter was much slower than in the leaves.
There is no evidence of a marked change in the rate of accumulation of
insoluble nitrogen in the stem at the time of flowering, although the
rate slowed down slightly. The soluble nitrogen of the stem likewise
accumulated slowly at first but, when rapid growth began, it increased
more rapidly than the soluble nitrogen of the leaves, overtaking this
at 61 days. Subsequently the soluble nitrogen of the stems was prac-
tically identical with that of the leaves, the changes being similar in each
case.

Growth of the Tobacco Plant 585

Nitrate Nitrogen: Figure 24 shows the quantities of nitrate nitro-
gen absorbed by the plant and stored as such in leaves and stem. More
nitrate accumulated in the leaves than in the stem during the first 54
days but, thereafter, the quantity in the stem exceeded that in the leaves ;
both curves show a steady accumulation of nitrate throughout the period
of rapid growth. At the time of flower and fruit development a sudden
diminution of the nitrate content in both leaves and stem began, and
this continued so rapidly that relatively little nitrate remained in the
leaves of the practically mature plants at 110 days. The stem, however,
still retained an appreciable quantity at this time. The behavior of the
nitrate suggests that, although absorption from the soil continued for
at least 97 days, as is shown by Figure 21, towards the end of the
growth period either the supply available in the soil was exhausted1 or
transformation of the nitrate into other substances then took place at a
rate that exceeded the rate of absorption. The rapid rise in the nitrogen
content of the developing pods suggests that the final stage of this trans-
formation consisted in the laying down of protein in the seeds.

Very little nitrate nitrogen accumulated as such in the developing
fruit ; in fact the quantities found represent little more than traces. This
suggests either that the nitrogen is translocated from the leaves into the
fruit in some form other than nitrate, or that the chemical transformation
of the nitrate in the pods takes place as rapidly as it is supplied from the
stem and leaves.

The behavior of the nitrate nitrogen of the tobacco plant is in marked
contrast with the behavior of nitrate- in the rhubarb plant as observed
by Culpepper and Caldwell (9). The nitrate nitrogen increased materially
in the petioles of their plants, so much so, in fact, that no less than 1.5
per cent of the dry weight of the senescent petioles might consist of nitrate
nitrogen. The leaf blades, on the other hand, did not greatly increase
their store, the proportion of nitrate nitrogen in all cases being less than
0.01 per cent of the fresh weight. Our data, when recalculated on the

1Mr. O. E. Street of the Tobacco Substation observed the nitrate content of the soil of a
field adjacent to that in which the plants of the present investigation were grown during the season
of 1933. The fertilizer applied to the two fields was essentially the same, but the crop studied by
him was Havana seed tobacco which is grown without a shade tent. Owing to the dry season, the
plants were irrigated on July 10 with the equivalent of 1.67 inches of water. The plants in the
shade field, used for our experiments, were irrigated in the same manner one week earlier, that
is, on July 3 and 4. We wish to express our thanks to Mr. Street for the following data.

Rainfall during

Time

Nitrate nitrogen

preceding interval

Date

clays

parts per million

inches

June 19

19

48.8

—

" 26

26

35.9

0.08

July 3

33

71.3

0.27

10

40

38.2

1.67 (Irrigated)

17

47

10.8

1.28

" 24

54

7.0

0.06

" 31

61

18.0

0.76

Aug. 14

75

69.5

1.19

21

82

3.0

0.28

Sept. 5

97

6.5

3.16

10

110

1.6

2.55

586 Connecticut Experiment Station Bulletin 374

basis of percentage of the fresh weight, show that the nitrate nitrogen
of the stems, which amounted to 0.14 per cent of the fresh weight at
the seedling stage, dropped in 40 days to the vicinity of 0.05 per cent
and there remained, with the exception of a sudden increase to 0.08 per cent
at 75 days, possibly due to the rain which fell the day before this col-
lection was made. The nitrate nitrogen of the tobacco leaf tissue started
at 0.08 per cent of the fresh weight and, with the exception of the 75-day
collection, dropped in almost linear fashion to 0.008 per cent at 110 days.

Nicotine Nitrogen : The quantities of nicotine nitrogen are shown
in Figure 25. The synthesis of nicotine began very early in the develop-
ment of the plant, and accumulation in the leaves proceeded rapidly through-
out the period of growth. The data indicate a drop in nicotine content in
the plants of the last collection in conformity with the drop of total nitrogen
that apparently occurred at this time. Nicotine accumulated slowly in
the stems throughout the period of rapid growth, but storage in the stems
ceased after 75 days.

Ammonia and Amide Nitrogen: The determination of amides in
plant tissue rests upon a difference in the stability of the amide nitrogen
of asparagine and of glutamine to hydrolyzing agents. The amide nitro-
gen of pure glutamine is quantitatively split off as ammonia when gluta-
mine is heated for two hours at 100° in a solution buffered at pH 6 to 7,
whereas the amide nitrogen of asparagine is scarcely affected under these
conditions. In order to hydrolyze the amide nitrogen of asparagine com-
pletely, it is necessary to heat the substance at 100° for three hours with
1 N sulfuric acid. Certain other substances behave in a similar manner,
and two of these, urea and allantoin, if present in substantial amounts in
a. plant extract, would make the accurate determination of asparagine and
glutamine by hydrolytic methods difficult, if not impossible. The deter-
mination, according to the methods we have adopted for the analysis of
tobacco leaf and stem tissue, involves the assumption that asparagine and
glutamine are the only amides present, and that no interfering substances
occur along with them. These assumptions are probably justified in the
case of tobacco leaf tissue. A careful search for allantoin in an extract
of tobacco leaves obtained from plants of the same type as those employed
in the present investigation failed to reveal its presence. The method em-
ployed was one that had succeeded in showing considerable quantities of
allantoin in an extract of tobacco seed, and it is felt, therefore, that the
quantity of allantoin in tobacco leaf tissue is probably so small that inter-
ference from this source with the amide determinations is not to be feared.
The absence of substantial quantities of urea is inferred from the o1is?r-
vation that the quantity of ammonia produced by hydrolysis of the tissue
at pH 7 for two hours is not increased by longer heating. Under the
conditions adopted for hydrolysis of glutamine amide nitrogen, only about
20 per cent of the nitrogen of urea is converted to ammonia; but further
conversion is brought about by longer heating.

The question of the presence of amides other than glutamine and aspar-
agine in the tobacco plant is, however, more difficult to answer. As will
be shown, the behavior of the tobacco stem tissue suggests the possible

Growth of the Tobacco Plant 587

presence of an unstable amide other than glutamine. In view of this it
is necessary for the present to employ the terms "glutamine amide nitro-
gen" and "asparagine amide nitrogen" in the following discussion in a
somewha#t broad sense ; although in certain cases there is reason to believe
that the two forms of nitrogen so designated are really derived from these
two substances, in others this is by no means certain ; consequently the
terms are to be understood to mean a form of nitrogen which behaves
in the same way as asparagine, or glutamine amide nitrogen respectively,
behaves.

That asparagine actually occurs in tobacco leaf tissue has been shown
by direct isolation (45) ; the presence of glutamine has not as yet been
confirmed in this manner. Notwithstanding this, there is no reasonable
doubt that the ammonia produced by hydrolysis of the dried tobacco leaf
tissue at pH 7 arises mainly from the amide group of glutamine. In fact
the determinations of glutamine in the leaf are probably intrinsically
more accurate than the determinations of asparagine, since the property
employed for the determination of glutamine is highly specific ; the as-
paragine determinations rest upon a difference between the free ammonia
and the acid hydrolyzable ammonia, and all amides are hydrolyzed under
the conditions adopted.

One further comment should be added before taking up the data in
detail. Asparagine, glutamine, and ammonia are extremely active metab-
olites in plant tissue. The proportions of the amides present are rapidly
influenced by changes in the proportion of ammonia in the plant, and
chance differences in the supply of ammonia from the soil in which the
individual plants grew would have an effect upon the amides. Irregu-
larities in the curves for the amides are therefore to be expected. These
irregularities are not to be regarded as errors ; they represent rather the
marked variability of the amides in response to conditions that could not
be controlled in a field experiment.

In discussing the data obtained, it is necessary to describe separately
the results from the two sets of samples taken at each collection. Figure
26 shows the quantities of free ammonia, of ammonia liberated by hy-
drolysis at pH 71, and by 1 N acid, in the dried leaves represented by
Sample II of each collection. The curves show a marked accumulation
of ammonia, and of amides, during the period of rapid growth, and in-
dicate that the quantity of free ammonia subsequently remained constant
for several weeks and finally slowly diminished. In Figure 27 the quan-
tities of glutamine and asparagine -amide nitrogen calculated from the
curves of Figure 26 are plotted. These curves indicate a slow but steady
accumulation of glutamine beginning from the 26th day ; asparagine ap-
parently did not begin to accumulate in substantial amounts until several
weeks later, but was then rapidly formed in considerable amounts. To-
wards the end of the experiment the quantity of asparagine diminished.

xTo avoid possible misunderstanding, it may be necessary to recall that the buffer solution
actually used was at pH 7.0. After being mixed with the tissue and heated for two hours the
reaction was in the range pH 6.3 to 6.6 in most cases. The exact reaction at which this hydrolysis
is conducted makes little difference so long as it is above pH 6.2 and below pH 7. In this connection
see Vickery, Pucher, Clark, Chibnall and Westall (in press).

588

Connecticut Experiment Station

Bulletin 374

Plotted on the same figure in broken line is a curve which represents
the "easily hydrolyzed amide nitrogen" of the leaves. This quantity is
obtained by subtracting the free ammonia of the dried leaf samples from
the free ammonia of the hot water extract made from the leaves of Sample
I of each collection. Granting that the leaves of Sample II and those of
Sample I are comparable, it represents the ammonia set free from some

0.06

0.04

0.02

0.00&

Pi*. 26

Leaves:

.Ammonia

and 7\

Amide N

o

fc/

HI

0 Days 40

80 120

0.06

0.02

0.00*

Fig. 27

Leaves:
Amide K

Days 40

0.10r

0.08

0.06

Fig. 28

0 Days 40 80 120

0.10

0.08

0.06

Fig. 29

Stems:
Amide N

0.02

0.00O ■ o-6

0 Days 40

unstable substance during the operation of preparing the hot water ex-
tract. Up to 47 days this curve is practically identical with that of the
glutamine amide nitrogen ; subsequently the agreement between the two
curves is not good, although they intersect each other in a manner that
strongly suggests that they represent what is fundamentally the same
thing. The reasons for placing more reliance upon the accuracy of re-
sults from the early collections have been discussed elsewhere, and there

Growth of the Tobacco Plant 589

seems little reason to doubt that the so-called "easily hydrolyzed amide
nitrogen" of tobacco leaves arises from glutamine.

The ammonia and amide nitrogen of the dried stem samples are plotted
in Figure 28. Free ammonia accumulated rather slowly in the stems,
.reaching a maximum of only approximately 10 mg. per plant in 61 days,
and thereafter diminishing slightly. The two forms of amide nitrogen,
however, mounted rapidly after the 35th day. In Figure 29 the aspar-
agine and glutamine amide nitrogen are shown. The curve for asparagine
amide nitrogen indicates, although with minor fluctuations, a steady ac-
cumulation of asparagine from 35 days to the end of the period studied.
Glutamine formed more rapidly at first, and appeared to reach a maximum
in 54 days that did not change materially thereafter.

The most striking feature of the diagram, however, is the curve for
the "easily hydrolyzed amide nitrogen". This factor obviously bears no
relationship whatever to the glutamine amide nitrogen of the stems as
directly determined, and the origin of the ammonia indicated presents a
difficult chemical problem. Discussion of this point will be deferred until
the data obtained from the extracts of the leaf and stem tissue have been
described. At the moment, attention is merely called to the curve which
represents the difference between the "easily hydrolyzed amide nitrogen"
and the glutamine amide nitrogen. This curve is designated "non-glut-
amine, easily hydrolyzed amide nitrogen". It rises rapidly to a well-
defined maximum at 61 days which is maintained until 97 days, and then
falls to a very low figure.

Figure 30 shows the free ammonia, and the ammonia produced by 1
N and by 6 N sulfuric acid hydrolysis, in the extracts from the leaves of
Sample I of each collection. If this figure is compared with Figure 26
it will be noted that, although the agreement is not entirely satisfactory,
the curve for free ammonia in Figure 30 corresponds roughly with that
for the pH 7 hydrolyzable ammonia in Figure 26, and that the curves for
the 1 N acid hydrolyzable ammonia nitrogen in the two figures are also
reasonably close. This is to be expected, inasmuch as the free ammonia
curve in Figure 30 represents the sum of the true free ammonia and of
the "easily hydrolyzed amide nitrogen" of the leaves, and should therefore
be the same as the sum of the free ammonia and glutamine amide nitrogen
shown in Figure 26. Similarly the 1 N acid hydrolyzable ammonia
nitrogen should be the same in each case.

The asparagine amide nitrogen of the leaf extracts is plotted in Figure
31. Comparison of this curve with that for the asparagine amide nitro-
gen of Figure 27 shows that the agreement in detail and in the general
behavior of the two curves is far from good. The analyses of the dry
leaf samples suggest that asparagine was formed only slowly in the early
stages of growth. The analyses of the extracts of a parallel series of
leaves, selected so as to be as nearly identical as possible, indicate that
asparagine was formed at a fairly steady rate from the earliest collections,
that it reached a maximum at 97 days and then diminished slightly. The
discrepancies between the analyses of these sets of samples clearly indi-
cate that the sampling error plays a very important part when dealing
with such highly reactive metabolites as the amides.

590

Connecticut Experiment Station

Bulletin 374

The difficulty that arises from the lack of agreement between the results
of the analyses of the two sets of samples is intensified by the extra-
ordinary results obtained from the stems. Figure 32 shows the ammonia

0.08

0.06

0.04-

0.02 ■

Fift. 30

Leaf Extract
Ammonia and
Amide N

**>
V

to/

r **/

V

7

Ik

PI ®

5 Days 40

80

120

0.08

Fig.

31

0.06

Leaf Extract:
Asp. Amide N and
6 N Acid Hyd.

Ammonia N

0.04

0.02

to
§
c

O.OOj

s*6

«*gF6 N Acid

)• Days

40 80 120

0.14

Fig.

32

0.12

Stem Extract: / I
Ammonia and / ■& I
Amide N J £• \

1 * \

0.10

r \

0.08

J// ©„ X

I \\

0.06

0.04

0.02

10

u
o

0.00<

5 Days

40

80

120

0.14

0.12

0.10

0.08

0.06

0.02

Fig. 33

Stem Extract:
Asp. Amide N and
6 N Acid Hyd.
Ammonia N

and 1 N acid hydrolyzable ammonia in the water extract from the stems,
and the solid line in Figure 33 shows the asparagine amide nitrogen
calculated from these data. It happens that the asparagine amide nitro-

Growth of the Tobacco Plant 591

gen so found agrees reasonably well with the data from the dry stem
samples plotted in Figure 29, particularly in the case of the younger
plants.

Another method to arrive at an estimate of the asparagine amide
nitrogen of the stem tissue is to subtract the sum of the free ammonia and
the glutamine amide nitrogen, determined in the dry stem samples, from
the ammonia produced by 1 N acid hydrolysis of the stem extract — that
is, to subtract the data plotted in the curve designated pH 7 in Figure 28
from the data designated 1 N acid in Figure 32. The results of this
calculation are plotted in the broken line in Figure 33. Clearly, there is
no relationship whatever between the asparagine so calculated and that
derived from analyses of the stem extract itself or of the dried tissue.
Mutually consistent results are obtained from each set of samples, but it
is not possible to employ data for the amides obtained from one set in
calculations of results from the other set.

Both the free ammonia and the 1 N acid hydrolyzable ammonia nitro-
gen of the stem extract are enormously greater than the analogous data
from the dried stem samples. In both cases the data are consistent, and
are fitted by fairly smooth curves ; the two sets of curves simply bear no
relationship to each other, in spite of the fact that they ostensibly represent
measures of the same thing. It should perhaps be emphasized that the
data in Figure 28, with the exception of the curve for free ammonia,
were obtained from analyses of hot water extracts of the previously dried
tissue, that is to say, the gross discrepancy between the data of Figures
28 and 32 is entirely a matter of whether the stem tissue was dried or
not before being extracted with boiling water.

It is apparently necessary to assume that the stem tissue contains a
substance which is decomposed with the production of ammonia, when
the tissue is extracted with boiling water at pH 4, and the extracts are
thereafter concentrated to small volume, and that this substance is con-
verted, during the process of drying the tissue, into a substance that no
longer gives off ammonia on being boiled with water.

It is of interest to inquire into the possibility that this hypothetical sub-
stance may be glutamine itself, that is, to assess the degree of proba-
bility that the glutamine determinations in the stem tissue may be very
seriously too low.

In the first place, the storage of glutamine in stem tissue in considerable
quantities under favorable conditions is a phenomenon that occurs in the
tomato plant (47) ; storage of glutamine in considerable amounts may
also occur in the fleshy root of the beet (unpublished observations).
Consequently one would be inclined to expect larger amounts of glutamine
in the stem tissue of the tobacco plant than in the leaf. The glutamine
data of Figure 29, if they are worth anything at all, indicate that this is
indeed the case (cf. Figure 27).

Glutamine is decomposed with the production of ammonia when its
aqueous solution is heated to 100°. The speed of the decomposition
varies with the reaction of the solution, being somewhat slower in the
vicinity of the isoelectric point (ca. pH 5), but decomposition is com-
plete at pH 7 in two hours. The other product of the decomposition is

592 Connecticut Experiment Station Bulletin 374

probably pyrrolidone carboxylic acid (8) as is shown by the diminution
of the free amino nitrogen of the solution. Pyrrolidone carboxylic acid
is converted into glutamic acid by boiling its solution for several hours
with acid of 1 N strength or greater. If, then, glutamine is present in
fresh tobacco stem tissue one would expect: First, that a loss of part,
or perhaps even all, of the glutamine should take place during the drying
of the tissue at 80 to 100°, particularly if this required a long time; second,
that a production of free ammonia equivalent to the glutamine decom-
posed in the drying tissue should occur; third, that a soluble substance
which yields amino nitrogen on acid hydrolysis should be demonstrable
in quantities commensurate with the loss of glutamine.

Unfortunately we have no data on the glutamine content of tobacco
stem tissue before and after being dried. Nevertheless, data to be pre-
sented in a forthcoming publication show that it is possible to dry slices
of the fleshy root of the beet plant in the same drying-oven equipment as
was used for the tobacco stems, with a loss of not more than from 10 to
15 per cent of the glutamine of the tissue as determined directly on the
fresh material. It therefore seems improbable that much of the glutamine
of the stems was destroyed during drying. This view is supported by the
data for the free ammonia of the dried stem tissue (Figure 28) ; at only
one point (61 days) was there as much as 10 mg. of free ammonia nitro-
gen per plant, whereas at this same time the glutamine determination
indicated the presence of 32 mg. of glutamine amide nitrogen, but over
80 mg. of easily hydrolyzed amide nitrogen. The order of magnitude of
the free ammonia nitrogen of the dried tissue, then, is not in accordance
with the view that any large proportion of the glutamine was destroyed
during drying, unless the somewhat improbable assumption is made that
the ammonia produced was driven out of the tissue at the temperature
of the oven.

It will be necessary to anticipate a little in order to correlate the possi-
bility of loss of glutamine during drying with the production of a sub-
stance that yields amino nitrogen on acid hydrolysis. In Figure 36 the
data for the peptide nitrogen of the stems are plotted. The observations
were made on hot water extracts of the dried tissue, and it should be
noted that the scale of ordinates is half that of Figure 29. Unfortunately
it is impossible to discriminate between actual peptide nitrogen and the
amino nitrogen resulting from the hydrolysis of pyrrolidone carboxylic
acid. It is clear, however, that, if the true peptide nitrogen of the stem
tissue is quite low, there is room for quantities of amino nitrogen arising
from pyrrolidone carboxylic acid of an order of magnitude comparable
with those shown in the curve for "non-glutamine easily hydrolyzed amide
nitrogen" in Figure 29. This argument, therefore, leads to the result that
it is possible that the determinations of glutamine in the stem tissue* are
serious underestimates of the quantity actually present. The argument is
weak, however, inasmuch as there is no good reason to assume that the
peptide nitrogen of the stems is in fact very low ; greater significance is
probably to be attached to the results of the free ammonia determinations
in the dried stem tissue.

Growth of the Tobacco Plant 593

We are therefore driven to the provisional assumption of a third source
of amide nitrogen in the stem tissue, and to the view that the curve desig-
nated "non-glutamine easily hydrolyzed amide nitrogen" in Figure 29
furnishes an approximate idea of the quantity present at each stage of
growth.

Manifestly the investigation of the amides of the tobacco plant is still
in a very elementary state. The methods of analysis hitherto developed
have validity only if certain assumptions regarding the chemical com-
position of the tissue are justified; that this is not the case with respect
to the stem tissue is evident, and it is possible that some doubt may be
properly attached to the results of the analysis of the leaf tissue as well.
If the present investigation does no more than to make this clear, the
labor expended will be well repaid.

6 N Acid Hydrolyzable Ammonia: In addition to the determina-
tions of the amide nitrogen by means of hydrolysis of the extracts from
the stem and leaf tissue with 1 N acid, a series of determinations was
made of the ammonia that is formed during hydrolysis for six hours with
6 N sulfuric acid. The chemical interpretation of this factor is, at present,
impossible. A part of it, at least, arises from the partial decomposition
of the purines, in particular adenine, known to occur in the plant ; part
may have some other origin such, for example, as the partial deamination
of amino acids. The data are presented merely for the sake of com-
pleting the picture of the various sources of ammonia nitrogen. Figure
30 shows the curve for the ammonia nitrogen produced by 6 N acid
hydrolysis of the hot water leaf extract, and the difference between this
curve and that for 1 N acid hydrolysis is plotted on Figure 31. The
quantity of ammonia nitrogen arising in this way closely follows, and is
nearly identical with, the quantity apparently present as asparagine amide
nitrogen. Figures 32 and 33 show similar data for the hot water stem
extract, and here again the behavior of the quantities involved closely
resembles the asparagine amide nitrogen.

Amino Nitrogen : The amino nitrogen of the leaf tissue, determined
upon hot water extracts of the dried leaf samples, is shown in Figure 34
by the curve designated "free NH2-N". The quantity present increased
very rapidly during the period of most active growth, and reached its
maximum at the time of blossoming; subsequently it diminished. Plotted
on the same diagram is a curve that shows the sum of the total glutamine
nitrogen and one-half the asparagine nitrogen. This curve should repre-
sent the quantity of amino nitrogen contributed by these two amides1,
inasmuch as glutamine yields approximately 90 per cent of its nitrogen
when treated with nitrous acid (8), and asparagine reacts only with its
amino group. The difference between the data of this curve, and those
of the free amino nitrogen curve, consequently represents the quantity of
amino nitrogen that arises from the amino acids themselves, irrespective
of the amides. The curve plotted from these differences shows an ac-
cumulation of amino acids, during the period of rapid growth, that pro-
ceeded to a maximum at 54 days and was maintained at this level for

1The assumption is made that no significant proportion of the glutamine was hydrolyzed during
the drying of the leaf samples.

594

Connecticut Experiment Station

Bulletin 374

three or four weeks. The period of development of the fruit, however,
corresponds with a rapid diminution of the amino acids of the leaves.
This behavior is strongly suggestive of translocation of amino acids as
such from the leaves to the fruit. In this connection it is interesting to
note that the sum of the glutamine and asparagine in the leaves did not

0.24

Fig.

34

Leaves:

Amino M

0.30

0.16

0.12

cq] \

0.08

n> nv / \
«>// o \

N 2 \

Nh \

0.04

mm / y\

a

B
m
u
c

hi *

w

o.ooi

) Day3

40 80 120

0 Days 40

120

0 Days 40

80 120

Fig. 37

Pods:

Amino and
Peotide N

0 Days 40

markedly diminish during this same period — a result that is contrary to
the view that these amides are of especial significance as translocatory
substances for nitrogen. Too much emphasis should not be placed upon
this result at the moment, however ; more comprehensive data than those
at present in hand will be needed to clarify the situation completely.

Relative Distribution of Constituents 595

In Figure 35 are plotted the results of the determination of the amino
nitrogen, after hydrolysis with 6 N acid, of an extract from the dried leaf
samples ; the curve is designated "total NH2-N". It represents the sum
of the amino nitrogen of the amino acids and amides already present
together with the amino nitrogen liberated by the hydrolysis of what are
presumably soluble peptides. Beneath it are plotted the results obtained
by subtracting the data for the free amino nitrogen. The curve shows
that soluble peptides accumulated but slowly in the leaves as growth
progressed ; from the 47th to the 97th day the quantity present did not
change significantly. There is evidence of a slight rise in the leaves of
the final collection ; in any case, however, peptide nitrogen does not ap-
pear to be an especially active component in the metabolic processes of
the leaves.

Figure 36 shows the free and total amino nitrogen determined in a
hot water extract of the dried stem tissue, together with the peptide
nitrogen. The free amino nitrogen, although always present in smaller
amount in the stems than in the leaves, rose fairly rapidly through the
period of rapid growth, was then maintained at a constant level for
several weeks and finally continued to rise through the period of blossom-
ing. The total amino nitrogen behaved in a manner closely like that of the
leaves ; it rose rapidly to a maximum at 75 days and then fell slightly.
The peptide nitrogen rose to a maximum at 75 days and then fell off
materially.

A calculation of the free amino nitrogen in the dried stems due to the
amides glutamine and asparagine leads to figures which are irreconcilable
with the data for free amino nitrogen — in every case quantities are obtained
that exceed the free amino nitrogen of the stems as directly determined.
This discrepancy furnishes a check upon the determinations of the two
amides in the dried stem extract which strongly suggests that the recorded
data for the amides are considerably too high and serves further to em-
phasize the inadequacy of the analytical methods for amides when applied
to tobacco stem tissue.

The amino and peptide nitrogen of a hot water extract of the dried,
fat-free pods are plotted in Figure 37. The rapid accumulation of pep-
tide nitrogen so that, in the last collection, the quantity was approximately
equal to that in the stems is of interest. It should perhaps be pointed out
that this peptide nitrogen does not represent the protein of the developing
seeds, as the protein of the seeds was not extracted by water under the
conditions adopted ; it probably represents soluble compounds of amino
acids synthesized in preparation for the laying down of the seed protein.

RELATIVE DISTRIBUTION OF THE CONSTITUENTS
OF THE TOBACCO PLANT

The analytical data hitherto discussed have for the most part referred
to the absolute quantity of each constituent, usually expressed in grams,
in a single plant. This method of presentation has furnished a picture
of many of the chemical details of the growth of the tobacco plant, but has

596 Connecticut Experiment Station Bulletin 374

given little direct information regarding the distribution of the various
factors in the different parts of the plant, or of the quantitative relation-
ships between these factors. It is customary to express analytical data
obtained from plants in terms of percentage of some quantity such as the
fresh or dry weight ; that is to say, the relative concentration of the
various factors is calculated in terms of a quantity which is assumed to
be constant. But, clearly, every quantity that might be employed as a
basis for the calculation varies as the plant grows, and one cannot assume
that the manner of variation of a given factor, and of the factor taken
as the basis of computation of the percentage, is in every case similar.
Irregularities in a curve of percentage may therefore be due to a change
in either or both of the quantities involved, and consequently become
difficult to interpret.

Accordingly, the calculations given below of the distribution of the
various components have been restricted for the most part to cases in
which the fundamental biological unit, the single plant, is still the dominant
factor in the situation, as our aim is to provide as vivid a picture as
possible of the chemical changes that occur during the growth of the
individual plant.

Figure 38 shows the distribution of the fresh weight of one plant as
between leaves, stem, and pods. The relative mass of leaf tissue increased
from 68 per cent to 85 per cent of the whole in 26 days after setting, but
then diminished fairly rapidly so that, at 61 days, the relative proportions
of leaf and stem tissue were approximately equal. The stem reached its
maximum relative proportion at the same time and thereafter remained
substantially constant, the diminution in relative weight of the leaves
being compensated by the increase in the relative weight of the blossoms
and pods.

The general distribution of the dry weight of the plant (Figure 39) is
very closely like that of the fresh weight, but there are differences in detail.
The dry weight of the seedling leaves was 77 per cent of the whole, being
10 per cent greater than the fresh weight, but, at 26 days, when it reached
its maximum proportion, it was only 2 per cent greater than the fresh
weight. The curves of dry weight intersect at 57 days, and thereafter the
proportion of dry stem tissue remained substantially constant, while the
pods increased and the leaves decreased. The lowest proportion reached
by the leaves was 27 per cent of the whole, being about 7 per cent less
than the lowest proportion reached by the fresh leaves.

Figure 40 shows the distribution of the organic solids and ash of the
plant. The curves for the organic solids, as might be expected, are closely
like the curves showing the distribution of dry weight. The curves for
the distribution of the total ash of the plant differ markedly, however, and
are plotted in broken lines on the same figure to permit close comparison.
The ash in the leaves at all times exceeded the ash in the stems, and only
in the leaves of the last two collections was the leaf ash as little as
one-half the total ash of the plant. Throughout the period of most rapid
growth more than two-thirds of the total ash was found in the leaves.

The distribution of the titrable organic acidity of the whole plant is
shown in Figure 41. The proportion of the total organic acids in the

Relative Distribution of Constituents

597

leaves rose from 82 per cent to nearly 94 per cent in the 26-day-old plants,
and then diminished almost linearly to 51 per cent in the leaves of the
last collection. The proportion of the acidity in the stems rose to about
30 per cent at the time of blossoming, and then remained constant during
the greater part of the period of pod development. Comparison of Figure
41 with the curves for the distribution of the ash in Figure 40 shows a
marked similarity between the two sets of curves. This furnishes another
example of the intimate relationship between the acidity and the ash con-
tent of the tobacco plant to which reference has already been made.

J38 Distribution of
Fresh Weight

59 Distribution of
Dry Weight

Days 40

80 120

Days 40

120

40 Distribution of
Organic Solids
and Ash

41 Distribution of
Total Acidity

0 Days 40

0 Days 40

It will perhaps have been noted that in every case considered the change
in distribution resulting from the development of the fruit has resulted
in a maintenance of the proportion present in the stem at approximately
the level attained at 61 days, while the proportion in the leaves diminished.
The distribution of the nitrogen of the plant, shown in Figure 42, striking-
ly illustrates this because of the high proportion of the nitrogen found in
the pods as these develop. The nitrogen of the leaves increased at the

598

Connecticut Experiment Station

Bulletin 374

start, reaching nearly 93 per cent of the whole at 27 days ; it then dimin-
ished fairly rapidly to 40 per cent at the last collection. Meanwhile the
stem nitrogen reached a maximum of only about 30 per cent at 61 days
and thereafter diminished slightly. The nitrogen of the pods, however,
increased with great rapidity to approximately 33 per cent at the final
observation, but the slope- of the curve suggests that the pods of still
older plants would contain an even larger share of the total nitrogen.

In Figure 43 are plotted two curves that show the relative proportions
of the total nitrogen of the leaves and stem respectively that are brought
into solution by boiling water. About 47 per cent of the nitrogen of the
seedling leaves was soluble, but this proportion rapidly dropped to a level
that varies between 25 and 30 per cent throughout the period of rapid
growth. The aging leaves of the last three collections, however, contained
a somewhat higher proportion of soluble nitrogen. This may perhaps
be associated with the fact that, during this period, transformations of the
leaf constituents into forms suitable for translocation to the developing
seed were taking place.

ioo r

Fig. 43 Distribution of
Total M

0 Days 40

00

rFig. 43 Proportion

of M

of Leaves

and

of Stems

as

Soluble

N

75

<>o-q Stems

50

<

\> Leaves^

^

25

C
d>

o

. M/^V

u
a,
0

3 Days 40 80

130

44 Distribution of

Nitrate and

Nicotine Ni

0 Days 40

The soluble nitrogen of the stems started at the very high proportion
of 82 per cent ; it dropped rapidly to 67 per cent, remained at this level
until rapid growth set in, when it dropped to about 63 per cent and there
remained until the last two collections when it fell a little further.

These two curves also furnish a rough measure of the variations in the
proportion of protein nitrogen in the two tissues. If the ordinates are
reversed and read downwards, the curves show the proportion of the
total nitrogen in each tissue that was insoluble in hot water. Much of the
insoluble nitrogen of the leaf tissue is certainly protein nitrogen, and it
seems probable that this is also true of the insoluble nitrogen of the stem.
It is clear that the proportion of protein nitrogen in the leaves rapidly
increased during the time the plant was establishing itself, but remained
relatively constant during the period of rapid growth. The proportion of
protein nitrogen in the stem likewise increased at the start, but remained
quite constant throughout the growth period ; towards the end there was
a minor increase in protein nitrogen.

Relative Distribution of Constituents

599

Figure 44 shows the relative distribution of the nitrate nitrogen in the
plant. The seedlings contained nearly as much of their nitrate in the
stem as in the leaf, but the relative proportion in the leaf rapidly increased
during the first 26 days. Subsequently it fell away so that in the final
collections nearly the whole of the nitrate of the plant was present in the
stem. The pods at no time contained more than a very small propor-
tion of the whole.

Plotted on the same figure is a curve which shows the proportion of
the total nicotine of the plant found in the leaves ; throughout most of the

[Fifi

. 47

Leaf N

as Amino

Per

cent

and

Peptide N

■

NH2-N

^Peptide

n/

Days 40

Days

0 Days 40

Per cent as Nitrate and
Nicotine N

0 Days 40

Days 40

Fi£. 50

Stem N
us Amino

Per oent

and

Pe

ptide N

^0

ID f
T3 1

a, I

K

2v

'mz-Tiw

0 Days 40

period studied this varied between 85 and 88 per cent. The constancy
of the proportion in the leaves is remarkable in view of the rapidly chang-
ing relative proportions of leaf and stem tissue as the plant grows, and
raises an interesting question with regard to the kind of cells that are
capable of synthesizing nicotine.

The distribution of the total nitrogen of the leaf among the various
forms that were determined analytically is shown in the next three figures.
Figure 45 shows the relative proportions of nitrate and nicotine nitrogen.

600 Connecticut Experiment Station Bulletin 374

The nitrate of the seedling leaves was very high, amounting to 23 per
cent of the whole. This rapidly diminished so that the nitrate in the
oldest leaves was approximately 2 per cent of the total nitrogen. The
curve shows several irregularities, notably at 35 and 75 days. Although
the possibility that these may be in part due to sampling error is not
excluded, it happens that each of these collections was made the day
following a change in the conditions of the culture of the plants. The
field was heavily irrigated on the 34th day, and on the 74th day the first
rain in several weeks fell. Under these circumstances it is perhaps not
surprising that a sudden increase in the nitrate content of the samples
taken on the succeeding days should have been observed. Although heavy
rain fell during the last week of the experiment, there was no response
in the nitrate content of the final collection, possibly because of exhaustion
of the available store of nitrate in the soil.

The proportion of nicotine nitrogen in the leaf increased very rapidly
during the first three weeks of the experiment, but then diminished slightly
and remained relatively constant during the early part of the period of
most rapid growth of the plant ; a steady increase in the proportion of
nicotine nitrogen then ensued and continued to the end.

Interpretation of these observations is difficult in view of our ignorance
of the exact position of nicotine in the scheme of nitrogen metabolism
of the tobacco plant. It may be suggested, however, that the synthesis
of nicotine was restricted, during the period of rapid growth, by the
demands of the tissues for nitrogen in other forms ; only when the plant
had attained a nearly full development was the synthesis of nicotine re-
sumed at a relative rate that resembled the initial rate of synthesis.

Figure 46 shows the relative proportions of three closely related forms
of nitrogen — ammonia, glutamine amide, and asparagine amide nitrogen —
in the leaves. These are highly reactive metabolites, and consequently the
curves which represent the proportions present are far from smooth. The
proportion of ammonia nitrogen fell rapidly during the period when
the seedling was establishing itself, and the curve of glutamine amide
nitrogen likewise descended. At 26 days the proportion of ammonia began
to increase and, by the expiration of 54 days, had reached its former level ;
subsequently it again diminished. Meanwhile the proportion of glutamine
amide nitrogen, after increasing slightly and diminishing again, remained
at a fairly constant level until at 75 days a marked increase occurred.

The curve of asparagine amide nitrogen shows that very little of this
amide was present in the young leaves, but that the proportion rapidly
increased during the period of rapid growth. The relationships between
the quantities of asparagine and of ammonia are in almost every detail
what is to be expected if the synthesis of asparagine is regarded as being
brought about by an increase in the free ammonia. Moreover, the de-
crease in free ammonia at the end may very probably be attributed to
transformation into one or the other, or perhaps both, of the amides.
These observations, then, are consistent with the view that amides are
synthesized by the plant as a protective measure.

The proportions of amino and of peptide nitrogen are shown in Figure
47. The data for the amino nitrogen are somewhat irregular, but indi-

Relative Distribution of Constituents 601

cate an increase of free amino nitrogen, during the period of most rapid
growth, to a level nearly twice as great as that at the start; this higher
level is maintained to the end. The peptide nitrogen, on the other hand,
remained, with minor variations, at a constant level of approximately 2
per cent until the last collection, when it suddenly increased sharply. This
change is probably associated with the rapid translocation of nitrogen
from leaf to seed pod that took place at this time.

The distribution of the nitrogen of the stem is shown in Figures 48,
49 and 50. The nitrate nitrogen in the tiny stems of the seedlings
amounted to no less than 65 per cent of the total nitrogen — an obvious
case of storage under what were designedly the most favorable possible
conditions for growth. During the period of establishment of the plant,
the nitrate diminished very rapidly indeed to the vicinity of 20 per cent.
Comparison of Figure 48 with Figure 45 shows that the stems also ex-
hibit the relatively higher proportion of nitrate at 35 and at 75 days that
characterized the leaves at these points — a further support for the ex-
planation already suggested.

During the period of rapid growth, nitrate increased in relative pro-
portion in the stem, but dropped in the stems of the last collection, possibly
due to leaching of the soil by the heavy rains of the last interval. At all
times, however, the nitrate made up a considerably larger part of the
nitrogen of the stem than it did of the nitrogen of the leaves.

The nicotine nitrogen of the stem is, save for the observation at 26 days,
relatively constant throughout at the low level of approximately 2 per
cent. In this respect the behavior of the stem nicotine contrasts sharply
with that of the leaf nicotine (Figure 45).

The curves in Figure 49 are presented purely with the object of com-
pleting the picture. No interpretation can at present be assigned to them
as the data represent the results of analytical determinations carried out
in routine fashion on the stem tissue in spite of the fact that these indirect
methods are probably not applicable in this particular case. Attention
should be directed to the fact that the rise and fall of the ammonia nitro-
gen is accompanied by analogous changes in the curve labeled "glutamine
amide N", but no direct inference of a relationship between these phe-
nomena is warranted until the chemical nature of the amide nitrogen of
the stem tissue is more fully known.

The proportions of amino and of peptide nitrogen in the stem tissue
are shown in Figure 50. The amino nitrogen dropped in the young plants
until rapid growth began, when the proportion increased. The low value
for the amino nitrogen at 75 days is difficult to explain and, with the
exception of this one point, the increase continued until the termination
of the experiment. The relative proportion of amino nitrogen in the stem
tissue is of about the same order. of magnitude as the proportion in the
leaf tissue.

The peptide nitrogen rapidly increased from a low value at the be-
ginning to a maximum approximating 8 per cent at 54 days ; subsequently
it fell to about 4 per cent at the end of the experiment. The behavior
of the proportion of this form of nitrogen in the stem is entirely different
from that in the leaf, and the possibility that some of the amino nitrogen

602 Connecticut Experiment Station Bulletin 374

produced by strong acid hydrolysis may have arisen from pyrrolidone
carboxylic acid has already been pointed out. Whatever its origin, how-
ever, the relative proportion is much greater in the stem, and the alter-
ations in this proportion, as growth progressed, are in striking contrast
to the constancy of the proportion in the leaf. If the peptide nitrogen of
the stem may be regarded as a measure of the quantity of partially con-
structed protein in transit from the leaves to the growing points, it is clear
that the maximal amount was present in the stem tissue during the period
of most rapid growth of the plant as a whole.

THE WORK OF SMIRNOV

Smirnov and his collaborators (35, 36), as has already been mentioned,
have also investigated the composition of the tobacco plant at various stages
in its growth. Their experimental technic and chemical methods differ
fundamentally in many respects from our own and, moreover, the variety
of tobacco plant they employed was likewise widely different, being a high-
carbohydrate, low-nitrogen type. Their data are expressed for the most
part in terms of grains per unit leaf area, and consequently there is no
way in which direct comparisons with ours can be made. Fortunately,
however, they likewise calculated the proportions of some constituents
in terms of percentage of the dry weight of the leaf, and these calculations
enable us to point out certain of the contrasts between the two varieties
of tobacco grown under widely different conditions.

An important consideration in a comparison such as this is the rela-
tive length of the growing season at Krasnodar and in Connecticut, and
the time required for the plants to reach maturity. It is difficult to corre-
late these factors. Smirnov refers to the leaves of his plants at 90 days
as technically ripe, while the 110-day-old leaves were beginning to turn
yellow. The lower leaves of Connecticut shade tobacco are regarded as
mature in about 50 days — in fact the first picking of the crop from the
plants in the field used in our experiments was made on the 51st day,
and the fourth and last picking had been completed by 75 days. But even
at 110 days of age the leaves of our plants had not begun to turn yellow.
This is an instance of the fundamental difference between the two types
of tobacco plant under discussion.

Smirnov's plants at 50 days had begun to form flower buds and at
63 days were in flower. A few flower buds had formed in the field from
which we drew our samples at 54 days, but flowering had not become
general until 61 days had elapsed. In view of this, the life cycle — at
least with respect to reproduction — is apparently not unduly different in
the two localities ; the great difference is in the use to which the re-
spective crops are put.

In Figure 51 is shown the percentage of total soluble carbohydrate in
the leaves of our samples of tobacco calculated on the basis of dry weight.
The curve drops sharply during the period of establishment of the plant,
rises as rapid growth began, but changes to only a minor degree through
the period of most rapid growth. As the plants reached their full size,

The Work of Smirnov

603

however, the percentage of carbohydrate rapidly increased to a high level.
Smirnov's data, calculated on the same basis, are plotted in broken lines
in the same figure. The soluble carbohydrate of his young plants rapidly
increased to a very high level, and then diminished again during the
period of rapid growth preceding blossoming. The curve for the topped
plants oscillates about a level of approximately 10 per cent. The behavior
of the two types of plant is thus quite different. Smirnov's plants at all
times, save in the 110-day collection, contained a much higher concentra-
tion of soluble carbohydrates than ours.

The proportion of nitrogen in the leaves of our plants, calculated as
percentage of the dry weight, is shown in Figure 52. The proportion
dropped slightly in the first period of study, doubtless clue to the rapid
metabolism of the large quantities of nitrate in the seedling leaves and the
increase in non-nitrogenous constituents, and then remained at a relatively
constant level of approximately 5 per cent until the plants were nearly
full grown. The level dropped in the leaves of the last two collections
very materially, probably due to withdrawal of nitrogenous substances
and translocation to the fruit.

Fig. 51 Leaf Dry flaight

Per c^nt as Carbohydrate
Smirnov •.*

opped

Fig. 52 Leaf Dry Weight:

0 Days 40

53 Leaf Fresh

Days 40

0 Days 40

Smirnov's seedlings were much lower in total nitrogen than ours, but
rapidly increased to the extraordinary figure of 6.2 per cent at 30 days ;
they then dropped again to a fairly constant proportion of about 4 per
cent during the period of rapid growth and beginning of flowering. Com-
parison of Figures 52 and 51 illustrates clearly the fundamental difference
in type of the two varieties of tobacco under discussion to which attention
has already been drawn. Interestingly enough, the topped plants studied
by Smirnov approached very closely to our own normal plants both in
nitrogen content, and in the behavior of the nitrogen in the aging leaves.

In Figure 53 the total nitrogen of the leaves calculated as per cent of
the fresh weight is plotted. This curve, unlike that of percentage on dry
weight, starts at a low level in the seedlings and rapidly rises as the
plants established themselves. During the early part of the growth period,
the proportion oscillated about a mean value slightly above 0.5 per cent,
and then gradually diminished to slightly less than 0.5 per cent. One of

604 Connecticut Experiment Station Bulletin 374

the values, that at 75 days, is extraordinarily high, the rest, however, are
remarkably constant. Smirnov's data, plotted on the same figure, indi-
cate that, when calculated in this manner, his leaves were richer in nitro-
gen than ours in all save the first case. He likewise encountered one
sample in the aging plants with an extraordinarily high nitrogen content.
Further investigation will be required to decide whether the aging leaves
do in fact pass through a stage of unusually high nitrogen content. We are
inclined, however, to attribute the high value in our own case to a sudden
influx of nitrate, due to the rain which fell after a prolonged dry period
during the 74th day of our experiment.

Further detailed comparisons of Smirnov's data with our own is per-
haps superfluous. Clearly the two types of tobacco are widely different
in their metabolism, and this is illustrated most forcibly by the difference
in nicotine content. The highest proportion of nicotine nitrogen attained
by our plants was slightly more than 10 per cent of the total nitrogen ;
Smirnov's oldest plants reached the astonishing figure of 34.2 per cent of
the total nitrogen as nicotine nitrogen. These plants had been topped
which, of course, profoundly altered the normal course of leaf metabolism,
and perhaps a comparison is not strictly justifiable. Nevertheless his nor-
mal plants at 63 days contained 16.3 per cent of the leaf nitrogen as nico-
tine nitrogen, whereas our 61 -day-old leaves contained only 4 per cent.

A few comparisons can be instituted on the basis of certain of the general
statements in Smirnov's paper. Thus he observed that the total carbo-
hydrate of the leaf tissue, when expressed in terms of surface area, in-
creased with age up to the time of flowering, diminished during the blossom-
ing period, and then increased again. Reference to Figure 16 shows that
the total carbohydrate of the leaf tissue of our plants, when expressed on
a gram per plant basis, underwent an analogous sequence of changes.

Smirnov noted the relative importance of oxalic acid in the young leaves
to which attention has already been directed. Unfortunately, however,
the methods he employed for the determination of malic and citric acid
are neither specific nor accurate, so that his conclusions with respect to the
behavior of these acids during the growth of the plant are not trustworthy.
Smirnov recognized this himself as he pointed out that the lack of suitable
methods of analysis made it impossible to obtain a clear picture of the
changes in the organic acids during the vegetative period of the leaves.

SUMMARY

The rate of growth of the tobacco plant has been investigated by means
of detailed chemical analyses of the leaves, stems, and fruit of a series of
collections taken at frequent intervals from the seedling stage to the point
at which the seed pods were well advanced in the process of ripening. The
variety studied was Connecticut shade-grown tobacco ; the plants were
grown under normal agricultural conditions under a shade tent as part of
a general crop.

The collections of plants were divided into two roughly equal lots, and
were then dissected into leaf, stem, and later, inflorescence, or pod por-

Summary 605

tions. The leaves and stems of one lot were separately extracted with
boiling water, those of the other lot were at once dried in an oven. The
pods were dried.

Analyses of the extracts, residues from extraction, and of the dried
samples were carried out by methods many of which have been specially
developed in this laboratory for application to the tobacco plant. The
results of the analyses were calculated on a basis of grams per individual
plant, and are plotted in the figures on a uniform time scale. Ratios between
certain of the constituents and distributions of some of them in the three
main parts of the plant have also been calculated.

The rate of growth, as measured by the increase of fresh weight of the
tops, was very slow during the first three weeks while the seedling was
establishing itself. The rate then rapidly accelerated to a maximum be-
tween the 40th and 47th days ; thereafter it diminished in a regular fashion
until seed production began. The relative proportions of leaf and stem
tissue changed profoundly during the growth period. At 35 days, the
stems weighed one-third as much as the leaves, but after 61 days exceeded
the leaves in weight. The leaves of the older plants diminished in weight
as the inflorescence developed.

Measured in terms of dry weight, the rate of growth was different in
many details. After an initial period of slow acceleration, the rate of growth
became practically constant in the period from 35 to 75 days, and sub-
sequently diminished more slowly than did the rate as measured in terms
of fresh weight.

Together with the results that show the broad aspects of the rate of
growth are presented data that show the rates of accumulation in leaves,
stem, and pods of the organic solids, the ash, the water-soluble organic
solids and ash, the total organic acidity, the quantities of malic, citric,
oxalic, and of unknown acids, the total and the fermentable carbohydrates,
the crude fiber, the ether extractives, the nitrogen both soluble and in-
soluble, and of the nitrate, nicotine, ammonia, asparagine amide, glutamine
amide, amino and peptide nitrogen. Where possible, attempts have been
made to correlate certain of the data with each other, and especial attention
has been given to the effect of the onset of the reproductive period.

It is not feasible to draw many detailed conclusions from a single experi-
ment that represents the effects of but one growing season. Attention may
be directed, however, to a few of the results that appear to have general
significance.

The data on the organic acids are the first that have hitherto been ob-
tained by accurate and trustworthy methods from which conclusions can
be drawn regarding the behavior of these substances in a growing plant.
A rapid accumulation of a high relative proportion of oxalic acid in the
very young tobacco plants was observed. This is a phenomenon already
noted by Smirnov and, if oxalic acid may be regarded as an end-product,
suggests an extremely rapid rate of metabolism in these plants. The most
significant result, however, is the observation that the three chief acids —
malic, citric, and oxalic — maintained a nearly constant ratio to one another
in the leaves from the 40th day to the end of the period of observation
(110 days), although the total quantity of organic acids present increased

606 Connecticut Experiment Station Bulletin 374-

about 400 per cent in the interval between 40 and 75 days and then sharply
decreased. This implies that the quantitative relationships of the three
acids to one another were not affected either by rapid deposition in the
leaves or by withdrawal from them. Manifestly the metabolism of these
three acids is closely related, and it is clear that oxalic acid shares pro-
portionately with the others in the chemical changes. No definite evi-
dence was secured, however, that connects the organic acid metabolism with
either the carbohydrate or the protein metabolism of the plant.

Malic acid was at all stages of growth the predominant acid of the
leaves, oxalic being the next in order, with citric in smallest quantity. The
amount of unknown acids was intermediate between the oxalic and citric
acids. In the stems, the unknown acids predominated, malic and oxalic
acids being present in considerably smaller amounts ; citric acid was in-
variably present, but in only small quantities. In the pods also, the un-
known acids predominated, malic acid coming next in quantity ; traces only
of oxalic and citric acids were present.

The investigation of the amide nitrogen showed that our knowledge of
the substances in the tobacco plant which produce ammonia on mild
hydrolysis with acids is far from complete. The results with the leaf
tissue could, for the most part, be satisfactorily interpreted on the assump-
tion that asparagine and glutamine are the only amides present. The
stem tissue, on the other hand, may contain a considerable proportion of
an unstable amide-like substance in addition to these ; if this is so, the
accurate determination of glutamine and asparagine by the methods em-
ployed is impossible.

The growth of the plant as a whole can be roughly divided into three
periods. The first is the period of from three to four weeks during which
the seedling established itself in the soil but increased little in weight.
The dry matter, organic acids, ash, carbohydrates and nitrogen in all forms
increased in absolute quantity per plant, but the relative distribution of the
individual organic acids, and of the forms of carbohydrate and of nitrogen
underwent considerable changes. Thus, the malic acid diminished, and the
oxalic and citric increased when calculated as percentage of the total acidity.
The proportion of the total soluble carbohydrate as fermentable carbo-
hydrate diminished. Calculated as percentage of the total nitrogen, the
nitrate nitrogen dropped profoundly, and the ammonia, amide, and amino
nitrogen diminished ; the nicotine nitrogen increased.

During the period of rapid growth, which extends roughly from the
35th to the 75th day, organic and inorganic substances accumulated in the
plant with surprising speed, but the alterations in relative proportions were
less striking. The individual organic acids were remarkably constant in
both leaf and stem. The proportion of fermentable carbohydrate in the
leaves dropped temporarily during the time of most rapid growth, but
soon recovered its earlier level, while the proportion in the stem steadily,
though slowly, increased. The relative proportions of the more active
nitrogenous metabolites, i.e. the nitrate, ammonia and amide nitrogen,
underwent material fluctuations, but in general the nitrate in the leaf
diminished, that in the stem increased. The nicotine of the leaves increased,
that of the stem remained constant, but the relative proportions of the total

Bibliography 607

nicotine in leaf and stem tissue were unchanged ; the amides and ammonia
of both leaf and stem increased.

In the final period, that of reproduction, which began at approximately
the 61st day, the leaves decreased both in fresh and in dry weight ; the
stems remained constant in fresh weight, but increased in dry weight ; the
organic acids and the ash of both leaves and stems increased, but the
relative proportions of the individual acids remained approximately steady ;
the soluble carbohydrates remained constant during the early part of the
reproductive period, but then increased. The total nitrogen of the plant
appeared to decrease somewhat at the end of the period of reproduction, but
additional evidence will be required to make this certain. The quantity of
nitrate nitrogen diminished sharply in both leaf and stem tissue ; the
quantity of nicotine nitrogen in the leaves increased, that in the stems re-
mained constant ; the amides and ammonia in general decreased in amount.
The relative proportion of nitrate decreased, that of the nicotine of the
leaves increased, while that of the stems remained constant ; the propor-
tions of amide nitrogen and of ammonia nitrogen increased.

The most striking feature of the final period was, of course, the evidence
of translocation of organic and of inorganic substances from other parts
of the plant, particularly the leaves, into the developing seed pods. Ap-
proximately one-fifth of the organic solids of the plant were ultimately
located in the fruit, and nearly one-third of this consisted of ether-soluble
material, mostly true fat. There was also a marked storage of nitrogen
in the seed pods, which at the end amounted to nearly one-third of the
nitrogen of the entire plant. Much of it was doubtless in the form of seed
protein. The translocation of organic acids, probably combined as salts of
inorganic cations, into the fruit was by no means a minor matter as about
one-tenth of the organic acidity was ultimately found therein. The picture
presented is very clearly one of transformation of the plastic materials of
the leaves and to lesser extent of the stems into the stable reserves of
nutriment for the succeeding generation.

BIBLIOGRAPHY

1. Assn. Official Agr. Chem., Methods of Analysis, 66. 1925.

2. Assn. Official Agr. Chem., Methods of Analysis, 117. 1925.

3. Barnstein, F., Landw. Vers. Sta., 54: 327. 1900.

4. Berthold, T., Conn. Agr. Expt. Sta., Bui. 326: 406. 1931.

5. Carpenter, F. B., N. C. Agr. Expt. Sta., Bui. 90a. 1893.

6. Chandler, W. H., Fruit Growing. (Boston) 1925.

7. Chibnall, A. C, Biochem. Jour., 16: 344. 1922.

8. Chibnall, A. C, and Westall, R. G., Biochem. Jour., 26: 122. 1932.

9. Culpepper, C. W., and Caldwell, J. S., Plant Physiol., 7: 447. 1932.

10. Davidson, R. J., Va. Agr. Expt. Sta., Bui. 50. 1895.

11. Emmerling, A., Landw. Vers. Sta., 24: 113. 1880.

12. Emmerling, A., Landw. Vers. Sta., 34: 1. 1887.

13. Emmerling, A., Landw. Vers. Sta., 54: 215. 1900.

14. Fleischer, E„ Arch. Pharm., 205: 97. 1874.

15. Gardner, N. R., Bradford, F. C, and Hooker, H. D., The Fundamentals of

Fruit Production. (New York) 1922.

608 Connecticut Experiment Station Bulletin 374

16. Garner, W. W., Bacon, C. W., and Bowling, J. D., Indus, and Engin. Chem.,

26: 970. 1934.

17. Gouwentak, C. A., Rec. Trav. Bot. Neerland., 26: 19. 1929.

18. Hartmann, B. G., and Hillig, F., Jour. Assoc. Off. Agr. Chem., 10: 264. 1927.

19. Jones, W. J. Jr., and Huston, H. A., Ind. Agr. Expt. Sta., Bui. 175. 1914.

20. Knowles, F., and Watkin, J. E., Jour. Agr. Sci., 21: 612. 1931.

21. Knowles, F., Watkin, J. E., and Hendry, F. W. F., Jour. Agr. Sci., 24:

368. 1934.

22. Koch, F. C., and McMeekin, T. L., Jour. Amer. Chem. Soc, 46: 2066. 1924.

23. Mason, T. G., and Maskell, E. J., Ann. Bot., 42: 189. 1928.

24. Peters, J. P., and Van Slyke, D. D., Quantitative Clinical Chemistry, 2:

385. (Baltimore) 1932.

25. Pucher, G. W., Leavenworth, C. S., and Vickery, H. B., Indus, and Engin.

Chem., Anal. Ed., 2: 191. 1930.

26. Pucher, G. W., Vickery, H. B., and Leavenworth, C. S., Indus, and Engin.

Chem., Anal. Ed., 6: 190. 1934.

27. Pucher, G. W., Vickery, H. B., and Wakeman, A. J., Jour. Biol. Chem.,

97: 605. 1932.

28. Pucher, G. W., Vickery, H. B., and Wakeman, A. J., Indus, and Engin.

Chem., Anal. Ed., 6: 140. 1934.

29. Pucher, G. W., Vickery, H. B., and Wakeman, A. J., Indus, and Engin.

Chem., Anal. Ed., 6: 288. 1934.

30. Roberts, W. L., and Schuette, H. A., Jour. Amer. Chem. Soc, 56: 207. 1934.

31. Schulze, B., and Schiitz, J., Landw. Vers. Sta., 71: 299. 1909.

32. Schulze, E., Ztschr. physiol. Chem., 24: 18. 1898.

33. Schweitzer, P., Missouri Agr. Expt. Sta. Bui. 9. 1889.

34. Sessions, A. C, and Shive, J. W., Soil Sci., 35: 355. 1933.

35. Smirnov, A. I., with collaborators, U. S. S. R. State Inst. Tobacco In-

vestigations, Bui. 46. (In Russian) 1928.

36. Smirnow, A. I., Erygin, P. S., Drboglaw, M. A., and Maschkowzew, M. T.,

Ztschr. wiss. Biol., Abt. E, Planta, 6: 687. 1928.

37. Sorensen, S. P. L., Biochem. Ztschr., 7: 45. 1908.

38. Stahl, A. L., and Shive, J. W., Soil Sci., 35: 375. 1933.

39. Stahl, A. L., and Shive, J. W„ Soil Sci., 35: 469. 1933.

40. Swart, N., Die Stoffwanderung in ablebenden Blattern. (Diss. Jena) 1914.

41. Thomas, W., Plant Physiol., 7: 391. 1932.

42. Tucker, G. M., and Tollens, B., Jour. Landw., 48: 39. 1900.

43. Vickery, H. B., Plant Physiol., 2: 303. 1927.

44. Vickery, H. B., and Pucher, G. W., Conn. Agr. Expt. Sta., Bui. 323. 1931.

45. Vickery, H. B., and Pucher, G. W., Conn. Agr. Expt. Sta., Bui. 324. 1931.

46. Vickery, H. B., and Pucher, G. W., Indus, and Engin. Chem., Anal. Ed.,

6: 372. 1934.

47. Vickery, H. B., Pucher, G. W., and Clark, H. E., Sci., 80: 459. 1934.

48. Vickery, H. B., Pucher, G. W., Wakeman, A. J., and Leavenworth, C. S.,

Chemical Investigations of the Tobacco Plant. Carnegie Inst. Wash.,
Pub. 445. 1933.

49. Vickery, H. B., Wakeman, A. J., and Leavenworth, C. S., Conn. Agr. Expt.

Sta., Bui. 339: 625. 1932.

50. White, H. C, Ga. Expt. Sta., Bui. 108. 1914.

51. Wilfarth, H., Romer, H., and Wimmer, G., Landw. Vers. Sta., 63: 1. 1906;

translated by Emslie, B. L., and published by Vinton and Co. (London)
1906.

APPENDIX

After the manuscript of the present bulletin had been prepared for the
press, we received the valuable papers of Vladescu (Vladescu, I., Buletinul
cultivarei si fermentarei Tutunului, 23: 231, 359. 1934) on the assimilation

Appendix 609

of mineral and organic substances during the development of the tobacco
plant. This extensive study, carried out at the Institute for the Culture
and Fermentation of Tobacco in Baneasa, deals with the composition of the
tobacco plant throughout the life cycle, particular attention being paid to
the inorganic constituents.

The plants studied by Vladescu were of the variety Molovata, a small-
leaved plant with slender stems especially suited for chemical investigations.
The behavior of these plants under the agricultural conditions that obtain
in Roumania differs fundamentally from that characteristic either of
Connecticut tobacco or the Russian variety studied by Smirnov at Kras-
nodar. After passing through the blossoming stage (46th to 56th days
from setting) the plants begin to lose in fresh weight owing to the ripen-
ing of the basal leaves (66 days). The ripening process gradually ex-
tends up the plant, and is accompanied by marked losses of water and
even of organic matter, possibly due to processes analogous to those noted
by the present writers during the curing of Connecticut tobacco leaves
(Carnegie Inst. Wash., Pub. 445: 1933). A renewed reproductive ac-
tivity begins at about the 96th day from setting and results in a distinct
increase in the fresh and dry weight of the plants as well as in the quantities
of inorganic constituents; but at the expiration of 119 days the fresh
and dry weight has again diminished.

Vladescu's papers deal explicitly with the matter of calculation of the
results of chemical analyses of plants during the growth cycle. He points
out that calculation in terms of percentage of the fresh or dry weight may
readily lead to contradiction and error. The calculation in terms of ab-
solute weight in a fixed number of plants (he uses 100 plants), however,
leads to definite and easily appreciated results, and is much to be preferred
for studies of growing plants.

His work is divided into two main parts. In the first part he deals
with plants taken from the seedbed at frequent intervals during the first
four weeks of vegetation. The plants had attained a size suitable for
transplantation in 17 days and at that time bore 4 to 5 leaves and were
12 cm. high. The entire plants, including the roots, were removed from
the beds, and washed free from soil ; superficial moisture was removed
and, after being weighed, the material was dried at 105°. The dry tissue
was then analyzed for total nitrogen, protein nitrogen, nicotine, and ash,
and the ash was analyzed for calcium, magnesium, iron, manganese, potas-
sium, phosphorus, and silicon by well known and accurate methods. The
results are expressed in tables and graphs which show both percentage of
dry weight and the absolute quantity in 100 plants.

Aside from the evidence of rapid assimilation of nitrogen and inorganic
substances, the most important observation is that nicotine is elaborated
at a constantly increasing rate even from the earliest stage, and forms a
very appreciable part of the non-protein nitrogen. It is unfortunate that
determinations of nitrate nitrogen were not included.

The second part of Vladescu's work deals with analyses of plants after
transplantation to the field. Four agricultural technics were employed in
the culture of these plants. Two equal areas of the experimental plot were
fertilized with manganese applied at the rate of 16 kilos of manganese

610 Connecticut Experiment Station Bulletin 374

sulfate per 1000 sq. m. in addition to the usual fertilizer. The suckers
were removed from half of the plants on each area at the proper time.
The final data therefore include observations on plants stimulated by the
administration of manganese, on normal plants, and on plants under both
conditions of nutriment from which the suckers had been removed.

The curves which • show the rate of accumulation, in the plants with
and without manganese, of fresh and dry weight, and of water, all indi-
cate a rapid rise to a maximum at approximately the 65th day from setting.
At this time the seed capsules were formed and drying of the basal leaves
had begun. During the following 30 days a diminution in weight occurred.
An increase to a second but lower maximum followed during the next
10 to 14 days, this second maximum being much more pronounced in the
case of the plants from which suckers had been removed.

The weight of the ash and, indeed, of several of the ash constituents
showed a similar, though in some cases less striking sequence of changes ;
iron, manganese, and silicon gave no distinct maxima, but the curves
reveal marked inflections at the critical points. Vladescu interprets these
changes in terms of positive and negative migration of the various con-
stituents, and points out that negative (downward) migration of iron,
manganese, and silicon apparently does not occur.

The behavior of the nitrogen in Vladescu's plants is of particular interest
because of the contrast to the behavior in Connecticut tobacco. The curves
as drawn in his paper indicate a rise to a maximum at about 65 days
(end of flowering) followed by a drop, and then a second rise to a sharp
maximum at about 108 days. The first maximum nitrogen content per
individual plant is 1.73 gm. which is very much less than the maximal
nitrogen content (5.12 gm.) of our plants attained on the 75th day. The
nitrogen of the plants then drops to 1.38 gm. at about 89 days, rises to
1.76 gm. at 108 days and finally drops sharply.

A careful study of the distribution of the points from which Vladescu
plotted his curve, and a consideration of our own difficulties in selecting
average plants so as to minimize the sampling error leads us to wonder if
Vladescu's data provide as clear a demonstration of a negative migra-
tion after the first period of flowering as he would imply. There is,
unfortunately, no information in his papers regarding the number of
plants taken at each collection and the magnitude of the probable error
cannot, therefore, be assessed.

Vladescu's data on the nicotine content of his plants, however, give
evidence of changes far greater than any possible experimental or sampling
error. The nicotine content reached a maximum at 65 days and then
steadily diminished to a value at 100 days of less than half the maximum.
The normal plants, that is, those from which suckers had not been re-
moved, then increased materially in nicotine, doubtless due to the elabo-
ration of alkaloids in the newly developing parts ; the others, however,
maintained the nicotine level unchanged through this period.

The quantities involved indicate that at the point of maximal nicotine
content (65 days) Vladescu's plants contained 0.0511 gm. of nicotine
nitrogen per plant of fresh weight 610 gm. Our plants at 61 days con-
tained 0.113 gm. in a plant of 954 gm. fresh weight. But at 61 days our

Appendix 611

plants had only begun to pass into the blossoming phase ; at the period
when blossoming was over and seed pods were developing (75 days), our
plants contained 0.182 gm. of nicotine nitrogen in a plant of 1,110 gm.
fresh weight, and subsequently the nicotine nitrogen increased to 0.287
gm. (97 days) with only a minor increase in fresh weight.

No phenomenon at all analogous to the profound drop in nicotine
content (from 0.051 gm. at 65 days to 0.028 gm. at 100 days) noted by
Vladescu occurred in our plants, and this is of great importance in the
interpretation of the physiological function of nicotine in the plant. We
have elsewhere (Carnegie Inst. Wash., Pub. 445. 1933) pointed out that
the nicotine content diminishes slightly during the curing of detached
tobacco leaves. But the rapid decrease that occurred in the leaves of the
Roumanian plants indicates that the destruction of the alkaloid in the
drying leaves still attached to the plant is a much more striking phenom-
enon and suggests the occurrence of a series of chemical changes, the
interpretation of which might throw much light on the function of nico-
tine in the tobacco plant.

612

Connecticut Experiment Station

Bulletin 374

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