LEAF
STARCH:
ITS
ISOLATION PROPERTIES
H. A. SPOEHR (From (From
the
Carnegie
Institution Stanford
(Received
HAROLD
AN of
AND
Was hington,
University,
for publication, publication,
SOME
OF ITS
W. MILNER Division
of
Plant
Biology,
California)
July Ju ly 30, 1935) 1935)
Our knowledge knowled ge of starch ha has s been gained gained largely from a study of the properties and composition of this substance as found in the storage organs of plants, such as seeds, seeds ,ubers, and roots. Although Although its presence n leaves as a product product of photosynthesis, tha thatt is autochautochthonic starch, has h as been known for a long time, and its formation, dissolution, dissol ution, and some som e of its properties in situ si tu have been studied with great care y mean means s of the microscope, little is known concerning the the chemical properties of starch of of this origin. The presence (or absence) of, starch in leaves has usually usually been recognized by by mea means ns of the iodine iodine test, and som s ome e endeavors have have been made for its quantitat quantitative ive estimation on the ba basis sis of hydrolysis hydrolysis means mean s of amylase preparations or of acid, though these methods are usually subject to to serious ser ious error. In the leaves of man many y species, specie s, on illumina illu mination tion,, starch appe appears ars with extrao extraordinar rdinary y rapidity, but we still sti ll ha have ve little definite information concerning the first sugar which is i s formed in photosynthesis and from which presumably this starch is formed. There There is usually a vari variet ety y of sugars present in the leaf; in only relatively few cases cas eshav have e these been carefull investigated as to their structure. structur e. It ha has s been difficult to account for the presence in plants of sugars, such as a s the pentoses, rhamnose, nose, I-galactose, whether the they y are direct products products of photosynthesis si s or are produced in subsequent subsequen t metabolic reactions in the plant cells. In leaves the starch is present chiefl chiefly y in the chloroplasts, chloroplast s, and its normal appearance has therefore therefore been associated associ ated with the photosynthetic activi activity ty of these organs. organs. The starch granule granules s of the chloropl chlo roplasts asts do not have have the characteristic structure of ordinary dinary starch grains. The isolation isolat ion of starch from the chloro67
D o w n l o a d e d f r o m w w w . j b c . o r g b y o n M a r c h 1 5 , 2 0 0 9
680
Leaf Starc
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 growt 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 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 w 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 s found by Irving (5) and by Thoday (6) The leaves we e collected after they had been exposed to bright light on a wa m day. Exposed to the vapors of chloroform, the leaves rapidly became flaccid, and after an exposure of about 0.5 hour, they we e dried in a stream of air at about 50”. For complete drying this required from 2 to 24 hours, depending upon the thickness and nature of the leaf. The dry leaf material s 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 s then ground fine in a large pebble ill for hours. This facilitates the extraction of the leaf pigments, for
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H. A. Spoehr and H. W. Milner
681
apparently some of the pigments are closely associated with th 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 s then repeatedly extracted by shaking with ethanol at room temperature for 5 to 15 hours. The last portions of chlorophyll we e removed only slowly, and to accomplish this it s found advantageous to use ethanol and petroleum ether alternately in the last three or four extractions. It is interestin that microscopic tests for starch with the usual iodine method in number of ases 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 s then extracted with water at room temperature by shaking the leaf material with 10 times its weight of wate for 4 hour and separating the remaining solid material by means of the centrifuge. This s repeated ntil the wa h water almost colorless. With leaf material which contained large amounts of mucilaginous substances, it s found necessary to extract in this manner six to eight times, and in such cases it advantageous to use larger amounts of water. The removal of substances which are soluble in cold water, such as gums an salts, particularly those of calcium, facilitates the isolation of starch in a later step in this process and also reduces the impuritie in the final product. The cold wa er extracts contained no starch or only very small amounts. The starch s w extracted from the leaf material with hot water. Water, equivalent to 10 times the weight of the dry lea powder after extraction with the organic solvents, s added to the residue from the cold wa er extraction and the mixture heated in a boiling wa er bath for 1 hour, followed by heating for 1 hour a 120”. After cooling, the solid material s separated by means of the centrifuge, and the solution s filtered through extra sof filter paper supported by a sheet of silk on a Buchner funnel. With leaf material containing a large amount of starch, it
D o w n l o a d e d f r o m w w w . j b c . o r g b y o n M a r c h 1 5 , 2 0 0 9
Leaf Starch found advantageous to make a second extraction or to use larger amount of water When a solution of starch is subjected to ow 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, nown as retrogradation, e have found that the starch can be completely precipitated from solution if proper precautions are observed. In our earlier experiments very ow temperatures, -185” and -8O”, we e used for the freezing of the starch solutions, but later experience howed that the precipitation of mos of the starch could be achieved by keeping the solutions at - 8” for 3 or 4 days.’ By the use of lower temperatures the starch is precipitated 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 completely, the filtrate giving no color wth iodine and, after hydrolyss 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 solution. The determinations of starch e 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 s recovered in this manner in one freezing. It may be that the o 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 s extracted from the leaf material and precipitated by freezing w s separated from the remaining solution by means of the centrifuge and s then filtered on silk. Th preparations obtained in this manner e dried in vacua over calcium chloride and ground as finely as possible in a steel mortar. They we e then dried in vacua over phosphorus pentoxide to constant weight. The starch which was precipitated from solution by freezin redissolved very readily when the solution s heated to 100”.
D o w n l o a d e d f r o m w w w . j b c . o r g b y o n M a r c h 1 5 , 2 0 0 9
H. A. Spoehr and H.
. Milner
683
When, however, the separated starch w s thoroughly dried, it more difficultly soluble, and even after heating to 120“ with water, there remained from 3 to 18 per cent undissolved, although this s hydrolyzed with 1 per cent hydrochloric acid. In additio to this there was always present in the leaf starches a small an 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 s much less. In some cases it s largely silica, probably in part at least from the pebble mill. 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 preparative purposes. Examination
of Chloroplast
Starches
The examination of the starch preparations from different leaves included the following. Ash-This s determined by incineration of 0.1 gm. samples. Nitrogen-This s 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 we e made on the basis of the reducing powe of such solutions after hydrolysis with acid. This s done by use of Benedict’s solution and the centrifuge method of Spoehr (12). A carefully prepared sample of potato starch s used as standard. Special experiments with both cornand 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 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 hydrolysis. It s found that the starch content of a solution can be
D o w n l o a d e d f r o m w w w . j b c . o r g b y o n M a r c h 1 5 , 2 0 0 9
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 s found experimentally that 1 gm. of “standard” starch hydrolyzed under these conditions had a reducing power equivalent to 1.053 gm. of pure glucose. This factor of 1.053 s used to convert the starch content to glucose content in the case of the hydrolyzed solutions obtained from the leaf starches. This value of the glucose content s used in calculating the specific rotation of the hydrolyzed solutions. The leaf starch preparations on hydrolysis 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 chloroplas starch, the opt,ical rotation of the hydrolyzed solutions s determined. From these determinations the specific rotation s calculated 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” s determined after they had been centrifuged to remove insoluble material. The starch content of these solutions w s determined on the basis of their reducing power after hydrolysis with acid, as described. The concentration of these solutions s of the order of 0.5 per cent. The optical rotation of starch solutions is, however, not a very satisfactory property for identification, o that these values cannot receive much weight. The results of the analysis of the starches obtained from 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 phenylosazones 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 ll 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 starc
D o w n l o a d e d f r o m w w w . j b c . o r g b y o n M a r c h 1 5 , 2 0 0 9
H. A. Spoehr and H. W. Milner
685
which has not been so treated (Table II). Solutions of retrograded starch showed maximum reducing power after 4.5 to hours hydrolysis with 1 per cent hydrochloric acid, as compared with 3 hours for the “standard” potato starch. The longer tim required for hydrolysis of retrograded starches probably also accounts for the fact that the relative reducing power of the lea starches s never so high as that of the potato starch, for the TABLE Ana lysis
Source
of Starches
from
of starch
I
:L ,eaf Sources
V arious
Ash
Nitrogen
Potato Corn
(tuber) (seed)
after hydrolysis
I .-
per cent
per cent
1ff1;
-~ per
cent
degrees
degrees
0.252
0.00
100.00
+194.6
+53.9
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
+170.2
+52.4
+198.8
+52.2
+188.5
+51.2
+237.5
+52.3
+lQQ.O
+52.5
+185.3
+52.6
93.05* Linum
usitatissimum
1.550
0.22
93.80 94.55*
Hedera
helix
(old
leaves)
1.069
0.12
88.88 89.34*
‘I
“
(young
leaves)
0.813
0.15
38.41 89.13*
Stellaria
media
0.947
0.23
89.65 90.51*
Echinocystis
jabacea
5.727
0.40
87.58 92.90*
Malva
parvijfora
0.748
0.19
92.64 93.34*
* Corrected weight
of the
for original
ash
by
subtra ction
starch
samp le.
of
the
weigh t
of
the
ash
from
the
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” potat starch which had been frozen for 4 days, thawed, the starch redis-
D o w n l o a d e d f r o m w w w . j b c . o r g b y o n M a r c h 1 5 , 2 0 0 9
Leaf Starch
686
solved, that is, without any separation of the starch from the liquid f the original solution, and the reducing power determined of this redissolved starch. This solution also showed maximu reducing power after hydrolysis of 4.5 to 5 hours, as s the case with the leaf starches which had been obtained by retrogradation. II
TABLE
Per Cent Hydrolysis
of Starch
of Different Origins Kind
Time of hydrolysis
Retrograded
potato
Toba cco
Dried
loo a0
95.05
93.81 95.26 95.67
99.38
95.88
96.49
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
98.97
98.14
97.94
97.32
98.35
97.32
97.11
92.16 94.02 94.85 95.46 95.88 96.29 95.46
97.32 97.32 96.70 96.29
95.88
Analyses
kaf
90.93
96.49
TABLE
Comparative
of starch
got separated
2.0
and Methods of Preparatio
92.78 94.43 90.93 90.93 92.37 92.78
93.61
III
of Tobacco Leaf Starch Prepared and by Precipitation with Ethanol
by Freezing
Method [al; at
Source of starch
N;;E-
A&
---~~ per cent per
co2
[aI;
center cent degrees
“Standard” potato.. . . . . . . . . . . . . . . . <0.07 0.252 0.26 Tobacco leaf pptd. with ethanol.. . . 0.20 0.345 0.57 ‘I “ by freezing. . . . . 0.12 0.219 0.38
+194.6 +196.0 +197.5
maximum redwing POWT ltfter hydrolysis degrees
+51. +49.3 +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 o The purpose polyuronides, such as gums and peck substances. of the cold water extraction of the leaf material s to free it as much as possible from these substances in order to avoid contami-
D o w n l o a d e d f r o m w w w . j b c . o r g b y o n M a r c h 1 5 , 2 0 0 9
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 ow reducing power. Thus, for example, the gum obtained from the cold wa er 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 n 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). an analysis is given of the starch obtained from For comparison, leaves by precipitation with alcohol. The tobacco leaf material s treated in the manner already described. The hot wa er extract s divided into o portions; from one of these the starch w s 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 powe than the starch prepared by freezing and exhibited maximum reduction after hydrolysis for 3 hours (Table II). The second ris in the reducing power, after longer hydrolysis of the precipitate starch, may be ascribed to the hydrolysis of the contaminating substances which we e precipitated with the starch. BIBLIOGRAPHY
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