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(From the Carnegie Institution of Washington, Division of Plant Biology,
Stanford University, California)

(Received for publication, July 30, 1935)

Our knowledge of starch has been gained largely from a study of

the properties and composition of this substance as found in the

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storage organs of plants, such as seeds,tubers, and roots. Although
its presencein leaves as a product of photosynthesis, that is autoch-
thonic starch, has been known for a long time, and its formation,
dissolution, and some of its properties in situ have been studied
with great care by means of the microscope, little is known con-
cerning the chemical properties of starch of this origin. The
presence (or absence) of, starch in leaves has usually been recog-
nized by means of the iodine test, and some endeavors have been
made for its quantitative estimation on the basis of hydrolysis by
means of amylase preparations or of acid, though these methods
are usually subject to serious error. In the leaves of many species,
on illumination, starch appears with extraordinary rapidity, but
we still have little definite information concerning the first sugar
which is formed in photosynthesis and from which presumably
this starch is formed. There is usually a variety of sugars present
in the leaf; in only relatively few caseshave these been carefully
investigated as to their structure. It has been difficult to account
for the presence in plants of sugars, such as the pentoses, rham-
nose, I-galactose, whether they are direct products of photosynthe-
sis or are produced in subsequent metabolic reactions in the plant
cells. In leaves the starch is present chiefly in the chloroplasts,
and its normal appearance has therefore been associated with the
photosynthetic activity of these organs. The starch granules of
the chloroplasts do not have the characteristic structure of or-
dinary starch grains. The isolation of starch from the chloro-
680 Leaf Starch

plasts makes possible the determination as to whether this has the

same composition as starch from storage organs and whether
leaves of widely differing species and of varying habit of growth
exhibit differences in the composition of the autochthonic
starch. Experiments are also in process to determine whether the
starch which is formed in leaves when these are fed different sugars
is always of the same or of varying composition.

Method of Isolating Starch from Leaves

In order to obtain starch from leaf material in as large quantity
and in as pure form as possible, special precautions must be taken

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in handling the leaf material. Although many species of leaves,
after a period of illumination, contain relatively large quantities
of starch (as high as 40 per cent of the dry weight), under certain
conditions, this undergoes dissolution with surprising rapidity.
It is now well established that in many leaves the starch content
rapidly decreases with loss of water from the leaves, so that in the
wilted condition they contain very little or no starch, although
the precise causes of this phenomenon have not been clearly deter-
mined (l-3). We have found that this depletion of the starch in
leaves does not occur if the leaves have been frozen or treated with
chloroform. This is in agreement with an older observation by
Brown and Morris (4) that chloroforming leaves inhibits the
dissolution of contained starch. Moreover, treating the leaves
with the vapors of chloroform greatly accelerates the rate of dry-
ing, although it also causes some leaves to turn brown, as was found
by Irving (5) and by Thoday (6).
The leaves were collected after they had been exposed to bright
light on a warm day. Exposed to the vapors of chloroform, the
leaves rapidly became flaccid, and after an exposure of about 0.5
hour, they were dried in a stream of air at about 50”. For com-
plete drying this required from 2 to 24 hours, depending upon the
thickness and nature of the leaf. The dry leaf material was then
ground for a short time and sifted through a 60-mesh sieve. In
this way the stem and fibers are separated from the more fragile
material of the mesophyll, which contains most of the starch and
constitutes about 30 to 50 per cent of the leaf material by weight.
This material was then ground fine in a large pebble mill for 3
hours. This facilitates the extraction of the leaf pigments, for
H. A. Spoehr and H. W. Milner 681
apparently some of the pigments are closely associated with the
starch granules, and the leaf material can be completely freed from
the pigments only after thorough grinding. It is also probable
that solution of starch in a subsequent step in the process of extrac-
tion proceeds more easily if the material is finely ground (7).
The leaf material was then repeatedly extracted by shaking with
ethanol at room temperature for 5 to 15 hours. The last portions
of chlorophyll were removed only slowly, and to accomplish this
it was found advantageous to use ethanol and petroleum ether
alternately in the last three or four extractions. It is interesting
that microscopic tests for starch with the usual iodine method in a

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number of cases yielded negative results before the material had
been extracted with petroleum ether but after such extraction gave
a strong and rapid positive test. There is apparently some
substance in these leaves which prevents the blue coloration of
starch with iodine (unless large amounts of iodine are used) and
which is removed by extraction with petroleum ether.
The leaf powder was then extracted with water at room tempera-
ture by shaking the leaf material with 10 times its weight of water
for 4 hour and separating the remaining solid material by means
of the centrifuge. This was repeated until the wash water was
almost colorless. With leaf material which contained large
amounts of mucilaginous substances, it was found necessary to
extract in this manner six to eight times, and in such cases it was
advantageous to use larger amounts of water. The removal of
substances which are soluble in cold water, such as gums and
salts, particularly those of calcium, facilitates the isolation of
starch in a later step in this process and also reduces the impurities
in the final product. The cold water extracts contained no starch
or only very small amounts.
The starch was now extracted from the leaf material with hot
water. Water, equivalent to 10 times the weight of the dry leaf
powder after extraction with the organic solvents, was added to
the residue from the cold water extraction and the mixture heated
in a boiling water bath for 1 hour, followed by heating for 1 hour at
120”. After cooling, the solid material was separated by means
of the centrifuge, and the solution was filtered through extra soft
filter paper supported by a sheet of silk on a Buchner funnel.
With leaf material containing a large amount of starch, it was
Leaf Starch

found advantageous to make a second extraction or to use a

larger amount of water.
When a solution of starch is subjected to low temperature for
some time and is then allowed to thaw, it is found that the starch
has been precipitated in a fibrous mass (8-11). In this process,
known as retrogradation, we have found that the starch can be
completely precipitated from solution if proper precautions are
observed. In our earlier experiments very low temperatures,
-185” and -8O”, were used for the freezing of the starch solu-
tions, but later experience showed that the precipitation of most
of the starch could be achieved by keeping the solutions at - 8” for

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3 or 4 days.’ By the use of lower temperatures the starch is pre-
cipitated from solution in a denser mass than at -8” to -10”.
Thus, experiments with a 0.5 per cent potato starch solution, kept
for 3 days at -8O”, showed that the starch is precipitated com-
pletely, the filtrate giving no color with iodine and, after hydrol-
ysis with 1 per cent hydrochloric acid, yielding no reducing sugars.
In a solution containing 5.063 mg. of potato tuber starch per
cc., there remained 0.526 mg. of starch per cc. after the solution
had been frozen for 4 days at -8”. In an identical experiment
with 3.592 mg. of corn-starch per cc., 0.177 mg. remained in solu-
tion. The determinations of starch were made by the method
described later in this paper. The results demonstrate that, by
freezing, about 90 per cent of the potato starch and 95 per cent of
the corn-starch had separated from solution. In actual practice
75 to 85 per cent of the starch originally in solution was recovered
in this manner in one freezing. It may be that the two amylose
constituents are not precipitated in exactly the same proportion in
which they exist in the original starch and this would, of course,
affect the composition of the resulting product in this respect.
The starch which was extracted from the leaf material and was
precipitated by freezing was separated from the remaining solution
by means of the centrifuge and was then filtered on silk. The
preparations obtained in this manner were dried in vacua over
calcium chloride and ground as finely as possible in a steel mortar.
They were then dried in vacua over phosphorus pentoxide to con-
stant weight.
The starch which was precipitated from solution by freezing
redissolved very readily when the solution was heated to 100”.
H. A. Spoehr and H. W. Milner 683

When, however, the separated starch was thoroughly dried, it was

more difficultly soluble, and even after heating to 120“ with water,
there remained from 3 to 18 per cent undissolved, although this
was hydrolyzed with 1 per cent hydrochloric acid. In addition
to this there was always present in the leaf starches a small and
variable amount of insoluble material other than starch. This
material remained insoluble after hydrolysis with hydrochloric
acid; in the starch preparations from Stellaria and Hedera it
amounted to about 5 per cent; in the others it was much less. In
some cases it was largely silica, probably in part at least from the
pebble mill.

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The yields of starch from different leaves, in per cent of the dry
leaf material after extraction with ethanol and petroleum ether,
are as follows: Nicotiana tabacum, 37.73; Phaseolus coccineus,
13.39; Linum usitatissimum, 2.26; Hedera helix (old leaves), 2.23,
(young leaves), 1.49; Stellaria media, 2.23; Echinocystis fabacea,
2.65; Malva parvijlora, 0.89. It should be pointed out that this
method in its present form does not give a quantitative value of
the amount of starch in leaves but is of use primarily for prepara-
tive purposes.

Examination of Chloroplast Starches

The examination of the starch preparations from different
leaves included the following.
Ash-This was determined by incineration of 0.1 gm. samples.
Nitrogen-This was determined by Pregl’s micro-Dumas
method; the result obtained may vary by ho.05 per cent.
Reducing Power-Quantitative determinations of the amounts
of starch in solution were made on the basis of the reducing power
of such solutions after hydrolysis with acid. This was done by
use of Benedict’s solution and the centrifuge method of Spoehr
(12). A carefully prepared sample of potato starch was used as a
standard. Special experiments with both corn- and potato
starch showed that the maximum quantity of reducing sugar is
present after 2.5 to 3 hours hydrolysis at 100” in the presence of 1
per cent hydrochloric acid when the starch concentration is of the
order of 0.1 per cent. With 0.5 per cent starch solutions, 3.5
hours under the same conditions are required for complete hydrol-
ysis. It was found that the starch content of a solution can be
Leaf Starch

determined with an accuracy of fl per cent by this method. In

the analysis of the starches obtained from different leaves, the
reducing power, after hydrolysis, is reported in terms of per cent
of the maximum reducing power of hydrolyzed potato starch. It
was found experimentally that 1 gm. of “standard” starch hydro-
lyzed under these conditions had a reducing power equivalent to
1.053 gm. of pure glucose. This factor of 1.053 was used to con-
vert the starch content to glucose content in the case of the hydro-
lyzed solutions obtained from the leaf starches. This value of the
glucose content was used in calculating the specific rotation of the
hydrolyzed solutions. The leaf starch preparations on hydrolysis

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yielded colorless or only slightly colored solutions.
Optical Rotation-In order to gain information concerning the
nature of the sugars produced by hydrolysis of the chloroplast
starch, the opt,ical rotation of the hydrolyzed solutions was deter-
mined. From these determinations the specific rotation was cal-
culated on the basis of the factor for the quantitative conversion of
starch into glucose, as indicated above.
The optical rotation of the leaf starch solutions which had been
prepared at 120” was determined after they had been centrifuged
to remove insoluble material. The starch content of these solu-
tions was determined on the basis of their reducing power after
hydrolysis with acid, as described. The concentration of these
solutions was of the order of 0.5 per cent. The optical rotation of
starch solutions is, however, not a very satisfactory property for
identification, so that these values cannot receive much weight.
The results of the analysis of the starches obtained from a
variety of leaf sources are given in Table I. The specific rotations
of the hydrolyzed leaf starches indicate that d-glucose is the chief
product. This is also substantiated by the fact that the phenyl-
osazones prepared from the hydrolyzed solutions of the various
leaf starches all melted at 207-209”, and micro-Dumas nitrogen
analyses agreed with the value calculated for phenylglucosazone.
Bearing on the point that the leaf starches which had been
separated from solution by freezing all showed a lower relative
reducing power as compared with potato and corn-starch is the
fact that the process of retrogradation produces a product with an
apparently lower relative reducing power. This is due to the fact
that retrograded starch is more slowly hydrolyzed than starch
H. A. Spoehr and H. W. Milner 685
which has not been so treated (Table II). Solutions of retro-
graded starch showed maximum reducing power after 4.5 to 5
hours hydrolysis with 1 per cent hydrochloric acid, as compared
with 3 hours for the “standard” potato starch. The longer time
required for hydrolysis of retrograded starches probably also
accounts for the fact that the relative reducing power of the leaf
starches was never so high as that of the potato starch, for the
Analysis of Starches from Various :L ,eaf Sources

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1 1ff1;
Source of starch Ash Nitrogen I I after
.- -~
per cent per cent per cent degrees degrees
Potato (tuber) 0.252 0.00 100.00 +194.6 +53.9
Corn (seed) 0.077 0.00 99.59 +lQO.O +51.8
Nicotiana tabacum 0.124 0.08 96.43 +201.3 +51.4
Phaseolus coccineus 5.482 0.34 87.95 +191.2 +50.6
Linum usitatissimum 1.550 0.22 93.80 +170.2 +52.4
Hedera helix (old leaves) 1.069 0.12 88.88 +198.8 +52.2
‘I “ (young leaves) 0.813 0.15 38.41 +188.5 +51.2
Stellaria media 0.947 0.23 89.65 +237.5 +52.3
Echinocystis jabacea 5.727 0.40 87.58 +lQQ.O +52.5
Malva parvijfora 0.748 0.19 92.64 +185.3 +52.6

* Corrected for ash by subtraction of the weight of the ash from the
weight of the original starch sample.

continued action of the acid on the glucose formed in hydrolysis

also results in the destruction of this to an appreciable extent.
That the lower reducing power of retrograded starch is not due to a
fractional separation of the original starch into portions of higher
and lower reducing power in the process of retrogradation is shown
by the results given in Table II, third column. These values
represent the reducing power of a solution of “standard” potato
starch which had been frozen for 4 days, thawed, the starch redis-
686 Leaf Starch

solved, that is, without any separation of the starch from the
liquid of the original solution, and the reducing power determined
of this redissolved starch. This solution also showed maximum
reducing power after hydrolysis of 4.5 to 5 hours, as was the case
with the leaf starches which had been obtained by retrogradation.
Per Cent Hydrolysis of Starch of Different Origins and Methods of Preparation
Kind of starch
Time of Retrograded potato Tobacco kaf

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got separated Dried


2.0 96.49 93.81 90.93

2.5 98.97 95.26 92.16 92.78
3.0 loo a0 95.05 95.67 94.02 94.43
3.5 99.38 95.88 94.85 90.93
4.0 96.49 95.46 90.93
4.5 98.14 97.94 97.32 95.88 92.37
5.0 97.32 98.35 97.32 96.29 92.78
5.5 97.32 97.11 96.70 95.46 93.61
6.0 95.88 96.29

Comparative Analyses of Tobacco Leaf Starch Prepared by Freezing Method
and by Precipitation with Ethanol
[al; at
Source of starch N;;E- A& co2 [aI; redwing
ltfter hy-
per cent per centper cent degrees degrees
“Standard” potato.. . . . . . . . . . . . . . . . <0.07 0.252 0.26 +194.6 +51.6
Tobacco leaf pptd. with ethanol.. . . 0.20 0.345 0.57 +196.0 +49.3
‘I “ “ by freezing. . . . . 0.12 0.219 0.38 +197.5 +48.9

The relative reducing power of the starch obtained from the differ-
ent leaves is, therefore, not very much below that of the pure
potato starch which had undergone retrogradation.
Leaves contain considerable, though varying, amounts of
polyuronides, such as gums and peck substances. The purpose
of the cold water extraction of the leaf material was to free it as
much as possible from these substances in order to avoid contami-
H. A. Spoehr and H. W. Milner

nation of the starch extracted with hot water. The material

extracted with cold water, when hydrolyzed by the method here
employed, had a relatively low reducing power. Thus, for exam-
ple, the gum obtained from the cold water extract of tobacco leaves
did not reduce Benedict’s solution nor give a blue color with
iodine; but after hydrolysis it had a reducing power about 50 per
cent that of hydrolyzed starch. That the leaf starches obtained
by the method here described contained only traces of polyuronides
is shown by the amount of carbon dioxide produced from them on
treatment with 12 per cent hydrochloric acid by the method of
Lefevre and Tollens (3 3) (Table III).

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For comparison, an analysis is given of the starch obtained from
leaves by precipitation with alcohol. The tobacco leaf material
was treated in the manner already described. The hot water
extract was divided into two portions; from one of these the
starch was precipitated by freezing; from the other, by addition of
ethanol, the final concentration of the latter being 50 per cent.
The method of precipitation by ethanol produced a starch with
higher content of ash, nitrogen, and polyuronides. The tobacco
leaf starch prepared in this manner had a lower reducing power
than the starch prepared by freezing and exhibited maximum
reduction after hydrolysis for 3 hours (Table II). The second rise
in the reducing power, after longer hydrolysis of the precipitated
starch, may be ascribed to the hydrolysis of the contaminating
substances which were precipitated with the starch.
1. Molisch, H., Ber. bot. Ges., 39, 339 (1921).
2. Schroeder, H., and Horn, T., Biochem. Z., 130, 166 (1922).
3. Iljin, W. S., Planta, 10, 170 (1930).
4. Brown, H. T., and Morris, G. H., J. Chem. Sot., 63, 656 (1893).
5. Irving, A. A., Ann. Bat., 26, 1090 (1911).
6. Thoday, D., Ann. Bot., 27, 713 (1913).
7. Alsberg, C., Grilling, E. P., and Field, J., J. Am. Chem. Sot., 48, 1299
8. Maquenne, L., Bull. Xoc. chim., 29, 1218 (1903).
9. Malfitano, G., and Moschkoff, A. N., Corn@ rend. Acad., 161,817 (1910).
10. Ling, A. R., and Nanji, D. R., J. Chem. Sot., 123, 2666 (1923).
11. Baldwin, M. E., J. Am. Chem. Sot., 62, 2907 (1930).
12. Spoehr, H. A., Carnegie Inst. Washington, Pub. No. 887, 31 (1918).
13. Lefevre, K. U., and Tollens, B., Ber, them. Ges., 40, 4513 (1907). Dick-
son, A. D., Otterscn, H., and Link, K. P., J. Am. Chem. Sot., 62, 775