Leaf Growth Factors Citation Bonner, David Mahlon (1940) Leaf Growth Factors. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/cp01-0h46. https://resolver.caltech.edu/CaltechTHESIS:02132025-222534811 Abstract No abstract. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: (Biology) Degree Grantor: California Institute of Technology Division: Biology Major Option: Biology Thesis Availability: Public (worldwide access) Research Advisor(s): Haagen-Smit, Arie Jan (advisor) Went, Frits W. (advisor) Thesis Committee: Unknown, Unknown Defense Date: 1 January 1940 Record Number: CaltechTHESIS:02132025-222534811 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:02132025-222534811 DOI: 10.7907/cp01-0h46 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 17003 Collection: CaltechTHESIS Deposited By: Benjamin Perez Deposited On: 14 Feb 2025 18:32 Last Modified: 14 Feb 2025 18:32 Thesis Files PDF - Final Version See Usage Policy. 34MB Repository Staff Only: item control page

LEAF GROWTH FACTORS Thesis by David M. :Bonner In Partial Fulfillment of the Req_uirements for the Degree of Doctor of Philosophy CALIFORlHA I l'JSTITUTE OF TECHlifOLOGY Pasadena, Californi~ Foreword For his teaching, a s well as for his inva,luable a dvice and criticism, the author would like to express his a-pp recia,tion to Professor A. J. Haagen-Smit, with whom this work was carried out. To Profes s or Went, who originally suggested t h e problem of 11 Lea.f Growth Factors", ana_ wi t b. whom the preliminary experiments were done, t he author gives his sincere t han..1rn. terest and his many helpful His continued in- suggestions were greatly appreciated. The author is greatly indebted to Professor H. E. Hayward at whose instigation the histological examination was underte.ken, and whose aid and advice made possible its carry i ng out. The author wishes to t hank Profes sor J. van Overbeek for mmw helpful suggestions and Professor E. G. Anderson for permis- sion to use the facilities of the farm at Arcadia, California. For capable technical assistance t he author is greatly i ndebted to Messrs. L. Castle and J. Greene. Lastly the author would like to take th:i.s opp ortunity of expressing his ap:9 reciation to h is brother James, with whom the green plant tests were carried out, for h is continued encouragement and unfailing sup ply of good ideas. Table of Contents ***** * * Page Introduction 1 Object of the present work 7 Chapter I The study of leaf grm-1th leading to the evidence for a hormonal control 8 Chapter II Preliminary eJq)eriments 12 Chapter III The leaf test 15 Chapter IV Additional methods of assay for leaf growth factors 25 Chap ter V Nature of the growth resp onse 32 Chapter VI Units and Standards Chapter VII The activity of various extracts and natural :products in the leaf test Chapter VIII Chemical fractionation of pea diffusate Chapter IX Activity of pure substances in le:u growth 55 Chapter X The response of excised pea embryos, and excised immature leaves to known leaf growth factors. 72 Chapter XI The influence of known leaf growth factors on the growth of green plants 77 Chapter XII Discussion 89 38 Conclusions 104 Literature Cited 106 -1- Introduction That plants require hormones, or correlation carriers, was postulated years ago by Julius Sachs. However, it was not until 1928 that the first plant hormone was clearly shown by Went (109) to be a definite chemical substance. To avoid possi- ble confusion with animal mechanisms, , the plant hormones were later termed phytohormones. The realization that organisms in other than the animal kingdom require vitamins for growth and development came even more slowly. In 1901 Wildiers (115) clearly recognized that yeast needs special growth factors which he collectively termed 11 bios 11 • Linossier in 1919 (61) pointed out that molds need vitamin like substances. Bottomley (20,21) de- monstrated in 1914 that many plants appeared to need accessory growth factors which he termed 11 auximon.es 11 and which he further showed were organic in nature. The beginning of the twentieth century was however strongly mechanistic, and these suggestions seemed too vitalistic to be accepted by the workers of the time, who spent considerable energy in attempts to show that bios and auximone effects were due to inorganic substances. With the iso- lation of the animal vit~.Jnins, and of the first phytohormone (K8gl and Haagen-Smit, 1931)(49) impetus was given to the study not only of growth factor requirements for microorganismbut also of the individual organs of higher plants, which has led within the past five years to the realization that the auximones of Bottomley rested upon a sounder basis than was originally thought. From this study of the hormones and vitamins of bacteria, fungi -2- a.nd higher plants there has developed a gradual recognition that the chemical factor or factors limiting the growth not only of different organisms, but of different organs within a single organism vary considerably. It is possible to recite at the present time numerous chemicei substances known to be the chemical factor limiting the growth of one or more organisms. It, however, has also become increasingly' clear that this multitude of requirements does not indicate variation in the chemical factors controlling basic cellular processes, but rather variation in the ability of the different organisms to synthesize these factors. There is little evidence indica ting differences in the basic cellular process.e s between the various organs of a higher plant. Differences in growth factor requirements between the various organs, may therefore be construed as owing to differences in synthetic ability of the different organs. Julius Sachs (90) nearly sixty years ago, brought forward the first complete as well as modern theory of morphogenesis and correlation based in essence upon differences in growth factor requirements of different organs. Sach 1 s starting point was the fact that morphological differences between plant organs must be due to differences in their chemical composition. ences he reasoned must be These differ- present at the time of the organ's initiation, even though the chemical methods available in the days of Sachs were too crude to detect differences of such small magnitude. He clearly distinguished these morphogenetic substances from ordinary foodstuffs, i.e. those substances present in much greater abundance and out of which the bulk of the plant is made. He also realized that these substances nm.st be present at very great dilutions, and their potency thus out of all proportion to -3t ne ir concentra tion. The identificat ion of t hese su~')sta..'l.ces postulated by Sachs he. s, however, proceeded but slowly. T'.ae first of Sachs I substances to be recognized was the f a ctor concerned in stem elongation. The evidence for t h is substance came mainly through work on phototropism, since as shown by Charles Darwin (30) in 1880, the effects of light and gravity are perceived by the tip of the p lant, and. the effects of this perception are then transmitted to the lower regions which in tum react. :Boysen-Jensen (22-24) in the years 1910- 1913 showed that the effect of unilateral light illumination could be transmitted across a wound gap, which findi ng was soon confirmed. by Pa.al (77). Paal was further able to demonstrate that t h e tip must be the primary seat of a growth regulating center, postulating that growth of the coleoptile is controlled by the tip through the agency of a diffusible substance (78). The demonstration of this diffusible substance as a definite chemical entity that could. be freed of the plant had, however, to wait nearly ten years after the work of Paal (77,78). Went (108,109) in 1928 was t h e first to successfully obtain the active substance free of t he plant, and. show that t h is extract has the same growth regulating properties as the original coleoptile tip. Went through suitable application of the bend ing induced in Avena plants by unsymmetrical application of this active substance to the decapi tated Avena coleoptile also devised a practicable q~antitative bio-asse;y for t he substance in question, the Avena te s t. With a q_uantitative assay for t his substance, and with t he knowledge t hat it could be obtained fre e from t he p lant, its isolation -4was possible, the pure crystalline form being first obtained in 1931 by X8gl and Haagen-Smit (49 ), who suggested the name 11 auxin 11 for this crysteiline substance active in promoting stem elongation. Following its isolation K8gl, Ha.a.gen-Smit and Erxleben (50-52) soon elucidated its chemical structure. Recent work of Went (110) has indicated that auxin is not the sole limiting factor for stem growth. His experiments indicate that a second factor produced in the roots, which acts in conjunction with auxin must be present for stem elongation. The chemical nature of this root factor is at present unknown, and Went (110) has tentatively termed it 11 caulocaline 11 • :Biotin is known to occur in pea seeds (55), and it has been shown to have a very marked promotive effect upon stem growth in excised pea embryos (55). :Biotin is also known to occur(55 ) in roots, and has recently been shown to be synthesized in isolated roots (10). That biotin and caulocaline are probably not identical, however, may be seen in the fact that biotin is much more abundant in the leaves than in the roots (55,10). Estroneis known to have a marked promotive effect upon the growth of excised pea embryos (55), and upon green plants (91,8). That this promotive effect of estrom is primarily upon stem growth is indicated in that estrone is totally inactive in promoting growth of isolated roots (13), or leaves (table 11). follows: Stem growth factors might be summarized as auxin and caulocaline appear to be the primary factors limiting stem growth, while secondary factors include biotin and estrone. It was clearly shown by K8gl and Haagen-Smit (54) that a different group of growth factors is required for root growth. They showed that concentrations of auxin which promote stem elongation strongly inhibit root elongation, indicating that auxin it- -5self was not concerned in root elongation. In order, however, that the growth factor req_uirements of separate organs be successfully established it is necessary as pointed out in 1902 by Haberlandt (40) that the organs be grown entirely freed from the influences of other organs. In the early 1920 1 s Robbins (82,83) and Kotte (59,60) were able to g row a number of different isolated root meristems . . These workers noted that additions of yeast extract, meat extra ct, or similar substances, to t he medium of inorganic s alts, and sugar somewhat increased .the growth of these isolated roots. "Potentially unlimited" growth t hrough many subcultures was reported in 1934 by Wh ite ( 114) for tomato roots upon a medium of sugar, inorganic salts, and small amount of yeast extract. Thiamin, known to be present in yeast extra ct, was noted by K8gl and Haagen-Smit (55) to considerably increase root growth of ex- cised pea embryos. Bonner (9) and Robbins and Bartley (86) found that thiamin could largely replace the effect of yeast extract on the growth of isolated root meristems of pea and tomato and was in fact essential for root growth. Thiamin . alone, however, could not entirely replace yeast extract over a long period of ti me. Addicott and Bonner (1) found nicotinic acid neces sary in addition to thiamin for continued optimum growth of pea roots, and Robbins and "Schmidt (87) found vitamin B6 a necessary second factor for growth of tomato roots. Recent work of Bonner (10,13) has indicated that thia~in requirement for root growth is quite general, and that different roots if requiring secondary factors, require nicotinic acid, or vitamin B6, or even both. may be summarized as follows: Root growth f a ctor requirements t hiamin appears to be needed by most roots, while seco ndary individual requi rements include such -6growth factors as nicotinic acid and vitamin :86. Many other growth factor requirements of higher :plants might be cited. Flower initiation is known to be controlled through an unidentified diffusible chemical substance (92). Initiation of root primordia is controlled by auxin in conjunction with rhizocaline (98,112). There have been few investigations, however, on the growth factor requirements of that important plant organ, the leaves. That so few investigations have been carried out upon this subject may be due to the tacit a,ssuJnption that leaves containing chlorophyll are able with the aid of sunlight to synthesize all the f a ctors they may need. -7- Object of the present work The object of the present work is an atte~t to e~tablish the general nature and mechanism of the growth factor requirements of isolated leaves, with the relationship of such requirements to the growth f actor requirements of stems and roots. -8- Chapter I *** The study of leaf growth leading to the evidence for a hormonal control. Prior to 1935 few direct studies of the growth factor requirements of isolated leaves were made. that of Vyvyan (102), and Of the earlier work, only of Gregory (37) bears upon this problem. Vyvyan using a photographic method with which he could measure leaf growth directly without detaching the leaf from the plant showed that the presence of the cotyledons affected the growth of leaves of seedling Phaseolus vulgaris plants, and that the removal of one of the two first foliage leaves considerab]y, increased the growth rate of the remaining leaf. He unfortunate~y worked with plants gro~m in soil and in sunlight, thereby obscuring the importance of the cotyledons for leaf development in seedling plants, and was led to ascribe his effects as due to availability of 11 food 11 , ma.1dng no distinction between carbohydrates, and accessory 11 food 11 , i.e. growth factors. Gregory using a photographic method similar to that of Vyvyan studied the increase in leaf area of Cucumis sativus under varying conditions. Measurement of the QJ.o of leaf growth le,d him to suggest that there was formed in older leaves under the influence of light a special factor or factors necessary for the growth of younger leaves. of light is not inconsistent with a He states, 11 the action hypothesis of a master photo- chemical reaction inc1ependent of carbon assimilation leading to the formation of a substance directly involved in leaf expansion." He thus clearly differentiated between the effect of carbohydrates on leaf growth, and of other special substances formed in the light. A further interesting observation of Gregory is of a close relationship existing between root and leaf growth in barley. He found the growth rate of the two organs closely similar, which he ascribed as due to the roots deriving their carbohydrates from carbohydrate synthesized in the leaves, and the leaves in turn deriving their nitrogen supply from the roots. Impetus was given the problem in 1935 through the work of Avery (3), Avery, working with leaves of Nicotiana correlated growth of the midrib and probably of the larger lateral veins with the auxin concentration of the leaf tissue. He found the highest auxin concentration where greatest elongation occurred. Ey apply- ing auxin in lanoline paste to the midrib of Nicotiana leaves, Avery found the leaf bent awa:y from the point of application, thus exhibiting a typical auxin growth response. A similar application to the mesophyll was without apparent effect. Went and Thimann lll3) corroborated these findings of Avery showing that auxin influences elongation of the cells of the midrib, and lateral veins with no increas~ in mesophyll area. On the basis of these experiments Went and Thimann (113) suggested tha t is is advantageous to distinguish between growth of the vein, which c~m be increased by auxin application, and growth of the mesophyll which is independent of auxin. The direct demonstration of a definite growth factor controlling mesophyll growth was obtained by Went (110). of his evidence is as follows: about The nature A group of etiolated pea plants ten centimeters in length were divided into four sets. The plants of the first group were left intact, the plants of the second group had their root cut off just below the cotyledons, the latter being left intact. In the third group the cotyledons were cut off close to the stem, thus leaving the stem and roots intact, while the fourth had both the root and cotyledons removed. Went found that removal of the roots did not affect leaf growth, the -10- mean leaf surface of such plants being exactly the same as that of plants whose roots had been left intact. Removal of the cotyledons, however, leaving the roots, exerted a very marked effect upon leaf growth, the leaves in such treated plants having but half the area of untreated plants. Removal of both cotyledons and roots had no greater effect than removal of the cotyledons alone, which would be expected since removal of the roots alone was without effect. Went 1 s conclusion from these observations was, tha t in the cotyle- dons there is present a substance necessary for leaf growth of etiolated pea seedlings. For the sake of convenience, Went termed this substance, present in the cotyledons affecting mesophyll growth, 11 phyllocaline 11 i ntending that this term be used only until chemi cal names could be used to describe the substance or sub- stances in question. Although removal of the roots did not affect mesophyll growth (i.e. mean leaf surfac·e) Went (llO) found tha t it did affect leaf length (i.e. vein growth) as well as petiole growth. This effect was very simila r to the effect upon stem growth produced by removal of the roots, thus substantiating the earlier conclusion that the growth factor requirements of vein and petiole growth are closel y similar to those for stem growth. In this connection it is of interest to note Stauffert~ (97) conclusion that vein growth and mesophyll growth are phenomena which are differentially affected by genes. For the laciniate form of Chelidonium maj],lS it was concluded that t his character apparently does not reflect a morphogenetic gene but results merely in growth . a decrea sed mesophyll -11- Further evidence for the existence of phyllocaline was obtained by Went through the use of transplantation experiments in which the etiolated tops of various pea varieties were grafted upon a variety of different root stocks (111). He found that the results of such experiments are obscured to some extent by the fact that not only are the responses dependent upon the cotyledon bearing root stock. but also upon the specific reactivity of the top. The pea varieties used as root stock affected leaf growth, as well as petiole, stipule and stem growth; the growth of these various organs being affected differentially. Differences in the dry weight of the seeds of different varieties were found to be small and since the bulk of the dry weight is due to stored carbohydrate, these differences, Went (111) concluded, must likely be due to differences in stored amounts of specific growth factors, as for example in amounts of stored phyllocaline. The work that has been reviewed leads to the following conclusions: Vein growth and mesop~rll growth are controlled by different sets of growth factors, vein growth being auxin dependent, and mesophyll growth being auxin independent. In the cotyle- dons of pea there is present a substance or group of substances capable of increasing the growth rate of leaves in etiolated pea seedlings. Under the influence of light older leaves are able to synthesize a substance distinct from carbohydrates which is essential for the growth of younger leaves. -12Chr1.p ter II *** Preliminary experiments. Since it was known that in etiolated pea seedlings, leaf growth depends upon growth factors stored in the cotyledons, pea cotyledons were chosen as a source of leaf growth factors. usual plant extract has the serious ~ne disadvantage that hydrolysis of the active principal may occur, and many toxic substances be released. In view of this fact a 11 diffusate 11 similar to that used by Xt3gl and Haagen-Smi t (55) in the :preparation of biotin from pea seeds was used . in preparing a crude extract of leaf growth factors. Since growth of the mesophyll under suitable conditions will be reflected mainly in increase in surface area, e:,q)eriments on growth in leaf area may be used to analyze growth factors of the mesophyll. It was found that a diffusate of pea seeds, as described above, caused appreciable growth in detached growing leaf sections, and that this growth could be simulated, in part at least, by the use of sugars and amino acids as the culture solutions. Early experiments and observations were carriecl. out with leaves of pea seedlings, and with the leaves of Carie~ p_a:eaya. To determine whether the factors diffusing from the cotyledons were in any manner correlated with the effect of these same cotyledons on leaf growth in the intact plant, Alaska pea varieties were compared. diffused, and the diffusates diffusates of Daisy and Eq_ual weights of peas were concentrated to eq_ual volumes. They were then tested for effect on leaf growth of Little Marvel peas, which had been found to show the greatest differences in leaf size when grafted on various varieties in transplantation (111). Diffusate from Daisy was found far more experiments effective than -13that from Alaska. Th i s is in agreement with the observations that leaf growth is greater in shoots grafted on Daisy than in shoots grafted on Alaska peas (111). When leaf strips, cut from the leaves of Car:!._c~ E_?,_p_~ya are floated on the solutions, it was observed that growth the edges of these strips was greater than that in the portion of the strip. along middle A more uniform resp onse was obtainable by perforating the le aves with a fine steel brush. Many cells are killed and wounded by such treatment, but t nis allows easier penetration of the solution to the intact cells in all parts of the section, resulting in uniform growth instead of growth merely along the cut edges of the section. This effect, however, is dependent upon the kind of leaf, presumably upon the permeability of the lower epidermis, and will be discussed later in chapter IV. Another observation was made in connection with the effect of the solutions on the parenchymatous regions of the leaf. A differential growth rate between parenchymatous and vascular tissue would be expected to result in a characteristic bulging of the tissue. When sections containing a large unstretchable vein are floated on a pea diffusat e medium they show marked bulging of the intercostal regions, since the parenchyma is growing more rapidly than the veins, yet the size of the section is determined by the length of the vein. This is just the reverse of the effect ob- tained by 2UXin treatment, when the veins bulge out between tautly stretched mesophyll. If sections are used which have small veins, however, these will be stretched by the growing mesophyll, and since the size of the section is c1etermined by the growth of the parenchyma the leaf section will be plane . These observations mey be summarized as follows: -14(1) A crude source of leaf growth factors may be obtained by collecting the water in which pea seeds have been submerged. (2) Leaf sections having small stretchable veins can be used satisfactorily in analyzing for mesophyll growth factors. (3) There is a correlation betv,een the factors diffusing from the cotyledons with the effect of these same cotyledons on leaf growth in the intact plant. -15Chapter III lll!l!lk The leaf test. In order that an object, a leaf in this case, be satisfactory for us~ in a quantitative bio-assey, (1) it must be reactive to the substance in question, and (2) the avera~e of several leaves mu.st be uniform from test to test. The following list gives the rather wide variety of leaves satisfying the first requirement, these being young rapidly growing leaves. ]rassica oleracea L. Carica papaya L. Lactuca sativa L., var. capitata L. Lactuca sativa L., var. longifolia L. Ludwigia L. Nicotiana sylvestris Spegaz & Comes. Nicotiana tabacum L. Raphanus sativus L. (French ]reakfast) Phaseolus vulgaris L. (Kentucky wonder) That rapidly enlarging tissue is more sensitive than slowly growing tissue was further illustrated by determining the sensitivity of different portions of a young leaf of Nicotiana sylvestris. As shown in table 1, the proximal portion is the most sensitive, and Avery (3) has sho\-m it to be the most rapidly enlarging portion. Older leaves (6 cm. long) are found to be either only slightly sensitive, or as a general rule totally insensitive. In order to satisfy the second requirement, it must be possible to collect the leaves in such a way that the growth rate of the leaves used for different tests, and therefore of leaves collected at different times, is limited approximately to the same extent by the leaf growth factors of some standard crude extract containing leaf growth factors, as for exaraple pea diffusate. It was ea.rly noticed that by collecting young leaves from c·e rtain pea varieties and from Nicoti~ plants, some leaves would grow almost as well in sugar as in the pea diffusate, showing that their growth -16rate was limited prima.rily by sugar. On the other hand, so me leaves of the same plant grew little in sugar but a grea t deal in pea diffusate, showing that t heir growth rate was limited by some additional factor. Such leaves obviously do not meet t he second re- quirement just mentioned. The first foliage . leaves from seedling plants, however, might be expected to be much more uniform in their sensitivity since they are dependent for their growth f actors upon themselves . and the cotyledons, and do not receive similar factors from older leaves. Nicotiana This was found tob3 the case seedlings. ~~e in Ra~hanus and first foliage leaves of seedling radish and tobacco plants have been the only such leaves worked with extensively, but from the number of p lants (listed above) that respond to the pea diffusate, it might be concluded that the eff ect of the leaf growth substances of the pea diffusate is not species specific. It has been found that in order to obtain leaves that may be worked with satisfactorily, the plants must be grown in the light. Leaves from etiolated pea seedlings proved too small and curled to work with, and in the case of radish there is no epicotyledona ry development in plants grown in the dark. An attempt was made to cause leaves to become more deficient in leaf growth factors by allowing the p lants to remain in the da r k a short period before collecting the leaves, and thereby increase their sensitivity. However, no further increase in sensitivity could be ob- tained by allowing plants to rema in in the dark for 24 before collection. The length of the to 48 hours photoperiod under which t he plants were grown too was fou. r1d quite without effect upon the sensitivity of the leaves to crude pea diffusate. -17Raphanus plants were finall;s,r selected for the leaf test, since large quantities of the seed were readily obtainable, and it is possible to grow large numbers of plants in a short time. Nicotiana can be used in the leaf test though less conveniently, as has been shown in numerous experiments. The r adish variety "French J3reakfast 11 , obtained through the Ferry Morse Seed Company of San Francisco, Cali f ornia, was used. The plants are grown in the greenhouse under standard conditions of planting and nutrient. When the first foliage leaves are about 50 sq. mm. in surface area they are cut from the plant, the time after germination being about 16-18 days under the conditions at this laboratory. It was found necessary to adhere rigidly to these conditions, since with older leaves, and with second foliage leaves the uniformity of reaction is greatly diminished. Circula r disks of approximately 19.5 sq. mm. in area are cut from the leaves by punching the!ll out with a sharp cork borer. negligible. The variation in size from disk to disk is The disks are carefully washed in distilled water, and shaken for five minutes on an electric shaker t o ensure thorough mixing of all of the sections. Fifteen to twenty sections per dish are then placed in Syracuse dishes each conta ining 2 cc. per dish of medium. Thi s number of sections was found to be sufficient to reduce the variation in sensitivity from dish to dish so that a reproducibility within a single test of 2-3 per cent can generally be obtained. The dishes are placed in an incubator a.t 25° + 0.5 C. for 30 hours. This length of time was found to give a maximum difference in area of about t hi rty per cent between control sections and sections grown in standard p ea diffusate medium. Allowing the test to run for longer t han thirty hours was found undesirable, -18- since the difference in area was enhanced little and the medium becomes badly contaminated. Several types of direct measurements were attempted for mea.surement of leaf growth. Leaf strips were used, t heir width being measured under a binocular microscope with an eyepiece micrometer. These measurements were abandoned, however, since owing to the resistance to stretching of the larger veins the growth of the sections was rather irregular. In order to gain sufficient reproducibility for satisfactory quantitative work it was found necessary to use a considerable number of sections, making direct measurements of area increase very laborious. Two equally reliable methods for measuring the growth were therefore worked out and used: increased (1) determination of the total wet weight of the sections from a single dish and. (2) measurement of the total surface area of the sections from a single dish. The method of wet weight determination is the faster, and was therefore used in routine testing, while the alternative method may be used as a, check. as follows: The method of obtaining the wet weight is a.11 the sections from a single dish are placed upon a piece of blotting paper, taking care that the secti.ons are separated from one another. They are dried on this paper for about one minute, and a definite number then transferred to a small beaker of known weight, their weight then being determined to a tenth of a milligram. These procedures are done in a uniform manner, and the length of time for each weighing made constant so that the error from varying degrees of dryness has been shown to be negligible. For measuring the total surface area a photoelectric cell is used. The cell is arranged in such a manner that a film may be passed between it and a suitable light source directly above the cell. -19By such an arrangement, only light the film is caught by the cell. transmitt ed by clear area s in The galvanometer deflection is then a measure of the amount of clear area on the film. Applica- tion of t his principle for measurement of leaf section areas is as follows: tihe sections are first dried as before and arranged on a piece of thin glass plate in sets of four, with lines of demarcation between the sections from dHferent dishes. A sha,dow photograph of the sections is then made on a strip of 32 mm. positive film, using a very weak exposure and an alkaline hydroquinone developer to insure maxinrum contrast. is shown in figure 1. Such a strip of film An entire experiment of 20-30 dishes may be photographed on two strips of film, so that error from varying film density is minimized. In addition two or three standard areas (disks cut from a piece of steel rod) are photographed on each strip of film, so t hat they may be used to calibrate the measurements to the same zero point on the galvanometer for each individual film. The film is then passed over the photo electric cell with a direct light source above it. The cell catches only the light transmitted through the clear spaces on the film, these clear areas corresponding to the surface area of the original leaf sections. The galvanometer deflection is noted, and the di f ference between the readings i s a measure of the difference in surface area of the original leaf sections. By this method the measure of activity is indicated by the increase in total surface area of twenty leaf sections grown in the crude extract over that of suitable controls. The correspondence between areas as measured in t his manner, and the determination of areas by measuring the photograph under a microscope with an eyepiece micrometer is very go od as is shown in table 2. The agreement between the method of determination of t he wet -20- weight of the sections and of determination of their surface area is shovm in table 3. The test done in t his manner is independent of pH from a pH of 4.0 to a pH of 7 .0, table 4. :Below pH 4.o the medium becomes toxic, owing to the external pH changing the internal pH sufficiently to ca;use damage to the tissue. The test is light indepen- dent, but is dependent on temperature, the sensitivity being less at lower temperatures. This chapter may be summarized as follows , A quantitative bio-assay for fe.ctors controlling the growth of leaves is as follows: circular disks ca. 19.5 mm 2 in area are cut from young first foliage leaves of Raphanus. Twen t y such discs are floated on the solutions to be tested, and allowed to grow on these solutions for 30 hours at 25° C. The total wet weight of all 20 sections from a single solution is then determined by direct weighing in a standard manner. The total surface area may be determined by suitable application of a photoelectric cell. The test is pH and light independent, but is temperature dependent. -21- Table 1 *** The sensitivity of different areas from a single leaf of Nicotiana Sylvestris Section cut from Growth in width above identical sections grown in 1% sucrose solution. Apex of leaf . . 0.8 mm . Base of leaf. . 1.4 mm. Edge of leaf . o.4 mm. Center of leaf, including mid-vein . 0.5 mm. A very young leaf . . . . . . . . . . 1.8 mm. -22Table 2 *** A comparison of activities measured with a photoelectric cell, and of direct mea surement of the film under a microscope with an eyepiece micrometer. Sections from 1'Ticotiana leaves in Method Photo Direct Water 100 100 1% Sucrose I 111 110 l 153 150 0.1 143 142 1% Sucrose+ pea diffusate mgs. dry wt./cc. -23Table 3 *** Comparison of activities determined by wet weight determination, and measuring surface area with photoelectric cell. Sections from Rap~~::us leaves in Method Wet weight detn. Area detn. mgs.).16 sections: Relative: Galvanometer:Rela tive to water: deflection in:towater controls: arbitrary units:controls Water 42.8 100 20.3 100 1% Sucrose 50.3 117 23.4 115 1 63.5 149 30.8 151 0.5 62.2 145 30.4 149 0.2 61.5 144 29. 6 146 0.02 59.2 139 29 .o 143 1% Sucrose+ pea diffusate mgs. dry wt.fee -24Table 4 *** Effect of pH on the leaf test Sections in EH of medium Growth relative to sugar controls 1% Sucrose Unbuffered 100 II 4.o 100 Unbuffered 110 II 7.0 110 II 6.o 111 If 5.0 110 II 4.o 109 1~ Sucrose+ pea diffusate 10 mg . dry wt . / cc . I Figure 1 Film arrangement for determination of leaf section surface area: a) sections of radish leaves grown 24 hours in water; gro\tm in 1% sucrose; b) c) grown in pea diffusate medium plus 1% sucrose. -25Chapter IV *** Additional methods of assay for leaf growth factors As a check against the leaf test it seemed desirable to use some additional mode of assay for the growth factor reQuirements of leaves. The leaf test while convenient in that several extracts may be simultaneously ana_ rapidly tested, has certain disadvantages. Partially mature, green leaf tissue is employed, and such tissue might be expected to contain some of the factors under investigation. The growth factor response and re~uirements of young etiolated leaf tissue might, therefore, be different. Tests of two general types were used to some extent in an investigation of this point, (1) leaf growth in excised embryos, and (2) growth of excised immature leaves. Aseptic embryo cultures have been employed for many years. :Brown and Morris (25) in 1890 described the use of excised embryos in the study of the seed-embryo relationship in barley, and other early work i ncludes that of Hannig (43) and Dietrich (31). The usefulness of this techniQue in biochemical investigations has been often pointed out (81) or as has been shown by Tukey (99 ) it can be very succes sfully employed in the circumvention of embryo abortion. The observation that in general the growth of excised em- bryos upon medium containing the essential inorganic elements and suga r was far less than in the intact seedling, led B~~nner (26) to suggest that developing plants just as developing animals req_uire "accessory gro\'1th factors". That excised embryos do req_uire gro\'rth factors can be seen in the investigation of IC8gl and Haagen- -26Smit (55), who found that ad.ditions of biotin, thiamin or estrone to the medium markedly increased the growth of embryos, biotin affecting primarily stem growth and thiamin primarily root growth. Similar observations were made by Bonner and Axtman (12), who further observed pantothenic acid and ascorbic acid (14) to exert a marked growth promotive effect upon excised embryos. Leaf develop- ment, however, as noted by :Sonner and Axtman (12) was marked.ly less than that of the intact etiolated seedling, and was not greatly influenced by any of the added accessory factors. The techniq_ue used in the present work is the same as that used by other workers (12,14). Seed of the variety 11 Perfection 11 * was used in all experiments. The seeds were sterilized by a brief wash of two minutes in 95% alcohol, followed by a soaking in 0.1~; HgC1 2 . twenty minute The seeds following this treatment were al- lowed to soak in sterile water for six hours, which sufficiently softens the seed to permit easy excision of the embryo. following excision were transferred to 50 cc. The embryos Erlenmeyer flasks containing 15 cc. of medium made up in 1% agar. The composition of the basic medium used is sho~m in table 5. This medium is the same as the basic meclium used for isolated root culture (11,13) which was found highly satisfactory for the growth of embryos (12). The desired amount of extra.ct to be tested for leaf growth activity is added directly to this basic mediu.'ll. Four embryos per flask were found satisfactory. allowed to grow in the dark at 25° C. for three weeks. time the embryos were removed from the flasks, and They were After this the total wet *Obtainecl thru the courtesy of FerrJ Morse Seed Co., San Francisco, California. -27weight of the roots, stems, and. entire leaves (mesophyll plus veins) determined for all of the embryos (as a rule 20) grovm in flasks of similar medium. Table 6 shows the marked effect upon As leaf growth of additions of pea diffusate to the medium. leaf growth appears to be a function of the pe a diffusate concentration, it is possible that this method may be used. quantitatively, though this was not carried to completion in the present work. As will be discussed in a later section, however, the requirements for leaf growth in embryos , appear to be similar to the requirements found in the leaf test. The following technique was employed in the culture of isolated immature leaves. Pea seeds of the variety "Perfection" wer9 sterilized as described for embryo culture. Intact seeds were germin<".t ed and grown in test tubes upon sterile agar. Twelve days after germination the apical leaves were cut off using sterile technique, these etiolated le e,ves being then floated upon liquid medium contained in 50 cc. Erlenmeyer flasks. The same inorganic medium described in table 5 was used with the exception that a solution 1% in sucrose was used rather than the 4% sho~m in table 5. The desired extract was added directly to tne medium. Figure 2 shows a shadow photograph of such leaves after growing one month in the dark at 25° C. Similar results, table 7, have been obtained using young radish leaves, fro:n radish plants grown in a ma..'1ner si milar to that just described for pea leaves, but left in the light, as no epicotyledonary development ta.'k:es place in the dark. The usefulness of this latter method as a quantitative bio-assay has not been fully investigated. It should be pointed -28out, however, t hat t he response of isolated immature leaves to crude leaf growth factor preparat ions and to crystalline active substances is similar to the resuonse of t he leaf test, (see chapter X). ~ This chapter may be sumillarized as follows: (1) Leaf growth (mesophyll and veins) of excised pea embryos is increased by the addition of pea diffusate to the mediu.~. (2) Excised immature leaves of pea and radish cultured under aseptic conditions respond with markedl y increased growth to the addition of pea diffusate to the culture medium. -29Table 5 !ll!l!li!. Composition of nutrient medium used in culture of excj_sed pea embryos and isolated immature l eaves. Substance Concentration mg.Lliter Ca (No 3) • 4H 0 2 2 242 MgS04·7H 20 42 KH0 85 3 KCl 61 KH/0 4 20 Fe (c R o ) •H 0 2 4 4 6 3 2 1.5 Sucrose 4o grams Table 6 *** Effect of pea dif:fusate upon leaf growth of excised pea embryos. Mgs. wet weight per leaf Relative to controls Control (nutrient only) 19.0 100 Nutrient+ pea diffusate mg. dry wt./1 25.0 31.5 165 2.5 25.8 136 .25 19.4 102 Treatment -31Table 7 *** Growth of immature leaves excised from radish seedlings gro'im sterilely in the light, cultured 4 . weeks. Wet --weight of 20 leaves _T_r_e_a_tr_n_en_t__________________~(_i_n__~rams) Control (water only) 0.210 Inorganic salt medium+ 1% sucrose 0.835 Inorganic salt+ 1% sucrose medium plus pea diffusate, mg. dry wt./1 4o Toxic 20 1.085 Figure 2 Growth of j eaves excised from etiolated pea seedlings, cultured one month. Top row: in water; middle row: in inorganic salt medium plus l ;b sucrose; bottom row: in inorganic salt and 17& sucrose medium, plus 1% standard pea diffusate solution. -32Chapter V *** Nature of the growth response. Histological studies under the guidance of Dr. H. E. Hayward have been started on both Nicotiana tabacum sativa leaves. and Ranhanus Disks were grovm in a manner similar to those gro\'m in regular leaf test, but were notched with regard to known orientation of the midvein. Navashin I s solution under a tion of the fixative. a The sections were killed with slight vacuum to allow quick penetra- They were then dehydrated in alcohol and tertiary butyl alcohol, and finally imbedded in a beeswax paraffin mixture. The sections were cut transversely and longitudinally at a thickness of 15 ,.µ.. and were stained with a modified Flemming's triple stain. Preliminary studies of both Nic~~i_§Ula and _gapl~nus indicate that a gross enlargement in area ancl in thickness of the sections has ta.~en place. Before killing the sections, measurements of area were made under a microscope with an eyepiece micrometer. They showed a 20% increase in area of sections grown in pea diffusate over sections grown in sucrose. Measurements of thickness were made ai'ter mounting the sections. T11e thickness was unifor!illy greater (mea sured from epidermis to epidermis) in leaves grown in pea diffusate than in sucrose. It is of interest also that in radish and tobacco the increase in thiclmess was rather uniform throughout the length of the sections. This would indicate that the growth factors p enetrated uniformly through the epidermis, and that in these two cases lack of penetration was not a limiting factor, as mentioned earlier for Carica ~ya. -33The structure and development of the leaf of Nicotiana tabacum has been excellently studied by Avery (2). The general structure of the mature lamina of the leaf of Nicotiana tabacum is shown in figure 3. Avery found that cessation of both cell division and cell elongation of the various tissues shown in figure 3 occurs independently and at different times. Cell divisions cease in the epidermis first, and in the palisade last. Moreover, cell division in the epidermal cells is generally complete by the ti me the leaf is one fifth to one sixth its mature size. Increase in surface area is then brought about entirely by cell elongation. While cell di visior.s ceas e first in the epidermal cells, elongation continues in them until elongation has ceased in the other tis sues, elongation continuing after enlargement of the middle and lower mesophyll hB,s ceased. Since the two tissues ceased cell division at nearly the same time, cell enlargement of the epidermis for a period longer than that of the spongy parenchyma results in a strain of the spongy .parenchyma cells. This strain produced upon the cells of the spongy parenchyma eventually becomes sufficiently great to pull the cells apart with the forma tion of intercellular airspaces. The palisade which is the last tissue to cease dividing is loosened, or may be pulled apart by continued enlargement of the epidermis. ~ne points of particular interest in coP..nection with the present work are tha t cell divisions are normally complete when the led' is approximately but one fifth of its mature size, and that cessation of both cell division and cell enlargement occur at different times for t h e epidermal, palisade, and spongy parenchyma cells. This differential growth of the various tissues of the lamina is of particular interest in that it might indica te differences in growth factor requirements for these thre e tis e:ues. Histological studie s indicate a general trend towards a greater degree of cell enlargement in sections of both Nicotiana, and Raphanus grown in the pea diffusate than in sucrose, figur e 4. In many sections examined this enlargement was very pronounced in the cells of the epidermis. No evidence of cell divisions was ob- tainea. which, in view of the findings of Avery discussed earlier, is not surprising. The leaves selected for the test had probably completed or nearly completed their cell divisions, and would be induced to further divisions only under abnormal conditions as for example high light intensity (32), or high salt concentration (41.~). Good evidence for a marked increase of intercellular air spaces in the spongy parenchyma is found in sections grown in crude pea diffusate. In the sections examined, however, evidence for a loosening of the cells in the palisade was inconclusive. The formation of intercellu.lax air spaces in the spongy parenchyma, as described earlier, is controlled by cell enlargement continuing for a longer period of time in the epidermal cells than in cells of the spongy parenchyma . Finding a marked intercellular air space formation in sections grown in pea diffusate is, therefore, suggestive that cell enlargement of the epidermis and probably of the palisade is more strongly affected than of the spongy paxenchyma. Strictly speaking, therefore, the f actors present in pea diffusate are not complete leaf growth factors. They do not affect vein growth, nor do they a~parently markedly influence laminal spongy paxenchyma. They do, however, increase cell enlargement of the laminal epidermal cel ls , and probably of the palisade cells. It must be emphasized that the present evidence is not conclusive, and that only tentative conclusions as to the tissue specificity of the factors present in -35pea diffusate can be drawn at present. Th is section may be sUJIL11arized as follows: (1) Leaf s ections from leaves of Nicotiana tab a cum and lwphanus sat i va grown in pea diffusat e show a gross enlargement in area and t hicknes s over sections gro~m in medium containing sugar alone. (2) The increase in thickness of sections grown in pea diffusate is uniform throughout the l eng th of the section indicating that lack of growth factor penetration is not a limiting factor. (3) Histological studies indicate a general trend towards a greater degree of cell enl argement with the formation of intercellul ar air spaces in leaf sections grown in pea diffusate t han in the cont rol sections. (l.~) The formation of intercellular air spaces in leaf sections grovm in pea d.iffusat e might indica te t hat the effect is prima irly upon cell enlargement of t he epidermi s and palisade, rather than upon cell enlargement in the spongy parenchyma. Struc t ure of t h e Lamina of a N"icotiana t a.bacum Leaf' Photomicrograph of a portion of the lamina of a tobacco leaf, X 200 Figure 3 OWER EPIDERMIS SPONGY PARENCHYMA (inidclle and lower mesophyll) - - - - - - - - - - - - - - - - PALISADE (upp er mesophyll) UPPER EPIDERMIS Figure 4 Photomicrographs of t he lan1ina of Nicotian a tabacum l eaf secti ons cultured for 30 hours in cliff eren t solutions a- water b- 1% sucrose c-1% sucrose p lus p ea diff us a te Figure 4-a Transverse section of a tobacco leaf section cultured 30 hours in water X 200 ( Figure 4-b Transverse section of a tobacco leaf section cultured· , 30 hours in 1%sucrose X 200 Figure 4-c Transverse section of a tobacco leaf section cultured 30 hours in 1% sucrose plus pea diffusate X 200 Chapter VI *** Units and Standards. In order that different extracts or pure substances be compared as to activity in promoting leaf growth, it was to devise an arbitrary measure of activity. Sugars, as mentioned Figure 5 shows the earlier, proved to be active in the leaf test. activity concentration curve obtained for sucrose. ty-pe of sugar used plays an important role. necessary The particular ]bth sucrose and glucose have the effect shovm in figure 5•. M.annose and galactose on the other hand give no appreciable effectF wnich indicates that the effect of sugars is not merely an osmotic one. The diffusate by itself has a high activity, as shovm in figure 6. To remove the possibility of a sugar being the limiting factor in the growth reaction, a medium containing sugar was added to all extracts tested and the growth compared with that of sections gro\om on sugar alone. The sugar selected was sucrose, it giving a good reaction, figure 5, and as seen from ta~le 9 pea diffusate contains 40% sucrose cal- culated as percentage dry weight. Two per cent by weight of sucrose gives optinrum growth when sucrose is used by itself. By trying severe.l dilutions it was found that in the presence of pea diffusate optimum growth was obtained with a solution one per cent by weight in sucrose, figure 7. All extracts were tested in a medium consist- ing of 1% sucrose, and activity was measured in terms of the added growth beyond that of sections grown in the basic mediu.rn. In era.er that activities be co mpared from day to da.y it is necessary to have a solution of 11 st 2.ndard activity". fine such a solution as one diffusate per cubic It was found convenient to de- conta ining 10 mg. dry weight of pea centimeter of one per cent sucrose solution. This solution is tested in a standard dilution series, consisting -37of four dilutions from 50 to 2 per cent, figure 8. It has been found tha t i f the diffusat e is concentrated to a syrup and stored in the cold, standard solution preparations from this concentrated diffusate give a relatively constant activity over a period of 2-3 months. The standard solution itself, however, mu.st be made up fresh every other day from the concentrated diffusate, since after that time its activity becomes variable. That such a solution may be used as a standard is due to the fact that standard solution prepara tions made from concentrated diffusates which were prepared at widely different times but from the same variety of pea have nearly the same activity. In calculating activities of extracts it is necessary to select an arbitrary unit. One leaf unit (L.U.) was activity of a solution containing one tenth defined as the of a cubic centimeter of the standard solution; that is, one milligram dry weight of pea diff usate per cubic centimeter of one per cent sucrose solution. The leaf unit concentration of an extract is determined by plotting the percentage increased growth (growth above that of the sugar controls) against concentration in mg.fee. The concentration of the extract which shows the same activity as the standarc1 solution at a concentration of one mg. dry weight of pea diffusate per cc. of 1% sucrose solution is determined graphically. This concentrati on then gives the milligrams dry weight of extract per leaf unit. This chapter may be summarized as follows: (1) The basic medium used in the leaf test is a solution 1% in sucrose. (2) A solution of "standard activity" is defined as one conta ining 10 mgs. dry weight of pea diffusate per cubic centimeter of one per cent sucrose solution. (3) One leaf unit (L.U.) is defined as the activity of a solution conta ining 0.1 cc. of the standard solution. z 3 4 /4 Sucrose 0 Figure 5 Effe ct of su cro s e on l ec1..f g rowt h e xp r essecl as growt h a.bove g rowt h of s ections in water. /20 //0 100,___ _ _ _J___ _ _ _L __ _ __ _ _ J i _ _ /.o 2.0 .3.0 MGS./ / CC . Figur e 6 }Tif f ec t of pe2 diffusat e on l eaf grou t h . Con c ent ra,t ion of :r;iea. di ffus a.te in terms of mg . dry weight of uea. diffu.s2-te p er c c . of wate r. Growth in water alone = 100. 5 3 MGS./ /cc. Figure 7. Growth of leaves in pea diffusa.te with different concentrati ons of sucrose. Growth expressed as growth above growth of sections grown in appropriate sucrose concentration. /ZO 2 3 4 5 Mosfcc. Figure 8. Standard dilution series. Growth expressed as growth above growth of sections grown in 1~ sucrose solution. -38Chapt er VII *** The activity of various extracts a.nd natural products in the lea£ test. A wide variety of different extracts and natural products have been tested for activity in promoting leaf growth, as measured by the leaf test. The extracts and natural products may be classi- fied as follows: (1) seed diffusates, (2) urines, (3) leaf extracts, and (4) miscellaneous. The method used in obtaining a seed diffusate is as follows: a) Sterilization of the seed: 1. Wash the dried seeds 10-12 times in tap water. 2. Wash 6-8 times in distilled water. 3, Place in a container from which the diffusate is to be collected (a large separatory funnel has been found excellent for this purpose). 4. Soak 2 minutes in 95% alcohol. 5. Rinse 5-6 times with sterile (boiled) distilled water. 6. Soalt 30 minutes in 0.1% mercuric chloride. 7. Rinse 6 times with sterile distilled water. b) Obtaining the diffusate: After the above treatment the seeds are covered with sterile water and left 12 hours. The water is then drained off and replaced by fresh sterile water. The water that has been drained off is concentrated in vacuo at 30° C. Pea seeds treated in this manner remain sterile and the leaf growth hormones (as biotin indefinitely, 55) continue to be given -39off at a constant rate for a very long time. Even after a month of diffusing leaf growth hormones are g iven off at nearly the same ra.te as when the diffusion was first started. Diffusates active in the leaf test have been obtained in a fashion similar to that just described from corn, radish, and pea seeds. A comparison of their activities in leaf units (L.U.) per milligram dry weight is shown in table 8. active. Of these three diffusates, pea diffusate 'l'ras the most This might be expected from the fact that the seed coat of corn allows little diffusion, and radish probably stores very little leaf growth factor, the first foliage leaves appearing only in the light. It should be noted, however, that the diffusates from these three widely different plants are all active in causing an increa sed growth rate in the radish leaf. This lends ada.itional support to the view that the active principle, or principles in each of the three diffusates is probably the same, and that common growth factors govern leaf growth in a wide variety of plants. Cow, mare, and human urine have all been tested for activity in the leaf test. Their activities in comparison to pea diffusate are shown in table 8. Cow urine was found to be highly active in leaf growth, as measured by the leaf test, being approximately one hundred times more active than the standard pea diffusate preparation. Human urine, while less active than cow urine still possesses considerably greater activity than pea diffusate. Mare urine was the least active of any of t he urines tested, though still ten times more active than pea diffusate. No active extract has been obtained by water extraction of fresh ground-up leaf tissue. However, by placing the leaves intact in sterile water and allowing them to remain in it at a low temper- ature ( to keep do\,m infection) it is possible to obtain a leaf -4odiffusate that is active in the leaf test. An extract of greater activity can generally be obtained by allowing the leaves first to soak in ether for 1-2 days, thereby inactivating destructive enzymes. A water extract of these ether killed leaves is then made. From young palm leaves (Wa~~~~tonia filifera[ an exceedingly potent extract may be prepared by allowing the etiolated immature leaves to soalt in sterile water for a week, draining the water off, and concentrating this diffusate in vacuo. This diffusate was the only highly active leaf extract obtained. Many other leaf extracts have been prepared, viz, radish, tobacco, bean, cocklebur, etc. table 8. W'nile they all possessed activity, their activities were considerably lower than the activity of pea diffusate. Yeast extract and 11 Acme 11 beer were both found active in the leaf test, and both had activity greater than pea diffusate, table 8. A water extract of egg yolk was inactive, while a similar extract of egg white had low but definite activity in the leaf test. Fruit juices as a rule were found to have either very low activity , or to be totally inactive. Pea diffusate was selected from among these possible crude sources for the fractionation and isolation of constituents active in leaf growth. While it is true that pea diffusate is considerably less active than cow urine, pea diffusate is of particular interest due to t he fact that it is prepared from pea cotyledons, knovm to be rich in the na turi:tlly occurtmg leaf growth factors. -41This chapter may be slwmiarized as follows: a) Seed diffusates obtained from pea, radish, and corn were found active in the leaf test. b) Cow, mare, and human urine were found highly active in the leaf test. c) The water extract of young etiolated palm leaves was found highly active in the leaf test. Other leaf extracts, though active, possessed low activity. d) Yeast extract, active in the leaf test. 11 Acme 11 beer, and egg white were found -42Table 8 *** Activity of various extracts and natural products in the leaf test, expressed as leaf units (L. U.) per milligram dry weight of substance. Substance tested L.U./rng. dry weight Pea diffusate 1.0 Corn 0.2 diffusate Radish diffusate *** Cow urine 100.0 Human urine 20.0 Mare urine 10.0 Washingtonia filifera leaves 80.0 Radish) II l'H cotiana) II Xanthium) II Phaseolus ) II Daucu~) II Lactuca) If 0.1 *** Yeast extract 4o.o Beer (Acme) 15.0 Egg white 0.1 Chapter VIII *** Chemical fractionation of pea diffusate . A fractionation of pea diffusate has been carried out which has led to the recognition of several distinct classes of substances contributing to the activity of the crude material. All of the active constituents have not as yet been recognized however, due in part to the lack of specificity eYJJ.ibited by the leaf test, and in part to the multiple nature of leaf growth factor requirements. Table 9 shows a general analysis of the pea diffusate. The bulk of the diffusate is accounted for by carbohydra tes ancJ. ash, both of which are active in leaf growth. The activity of the car- bohydrate fraction can be duplicated and its effect obviated t:nrough the use of sucrose in the medium. The activity of the ash on the other hand contributes to the activit~r of the crude material. The magnitude of thi s contribution will be discussed later in t his chapter. Several di f ferent methods of fractionation have been used. It would be of little value to discuss those yielding inconclusive information concerning the nature of the growth factors. the two yielding the most Rather fruitful results will be described, using a typical fractionation as example. The fractionation schemes are shown diagrammatically in figures 9 and 10. I. Benzene ~~__P-etrol ether extraction: 95 grams of pea diffusate concentrated to a syrup was extracted in a continuous extractor for 24 hours with benzene, followed by 24 hours extraction with petrol ether (boiling point 30-L(Q). Initial analysis showed 9,000 L.U. present in the total sample. One -44gram of mat erial proved s oluble, with the entire activity rema ining i n the water fraction. Total dry weight of water soluble fraction - 95 grams Total number of leaf units - - - - - - - - -9,000 1.TJ. /mg. 0.1 II. Alkaline ether extraction: The extract was made alkaline with barium hydroxide and extracted with peroxide free ether on a continuous extractor for 48 hours. 200 milligrams of inactive ether soluble material was obtained. The barium was removed through precipitation as barium sulfate by adding an e~uivalent amount of sulfuric acid. This precipitation carried with it 10 grams of inactive material, and gave an increase in the total number of leaf units present. Total dry weight of water soluble fraction -- 86 gra...11s Leaf units - - - - - - - - - - - 30,000 Enrichment - 2.8 X. III. Acid ether extraction. The active water extract was made acid with sulfuric acid, and extracted with peroxide free ether in a continuous extractor. After extraction was complete the sulfuric acid was removed as bari"wn sulfate by ad~ition of barium acetate. The acetic acid formed in this treatment was then removed by continued extraction with ether. Water soluble Total dry weight Leaf units ... ..,. Enrichment 6X Ether soluble - - 85 grams - 50,000 Total dry weight - - - 1 gram. Leaf units - - - - 6,600 -45IV. Mercuric acetate _pI'.eC2£i tation The water soluble fra ction from t he preceeding step was fractionated with mercuric acet a te according to the method of Klein (47). The extract was maintained. slightly alkaline at all times during the precipitation by adding a lo% sodium carbonate solution. Mercuric acetate solution (25%) was added giving rise to a fine white precipitate. It was added until precipitation was complete as indicated by the formation of a bright red mercury complex. The precipitate wa.s filtered off, suspended in water and decomposed with hydrogen sulfide gas. The mercuric sulfide formed was filtered off, and the extract concentrated in vacuo. The excess mercuric acetate present in the mother liquor was removed in a similar fas hion, and the acetic acid formed during the original precipitati on was removed by ether extraction. Mother ~<?.!. Pre~ipitate Total dry weight Leaf uni ts - - 26.5 grams. Inactive. - - 30,000 Enrichment - llX The water extract obtained from the above precipitate was allowed to remain in the ice box for sever al days. A fine white precipitate formed which was filtered off, and was recrystallized from alcohol-water as the potassium salt. This substance was found to have a low activity in the leaf test, giving a maximum increase in wet weight of 101i . Elementary analysis gave: *Microanalysis by G. A. Swinehart. * -46- FoW1d %c CbH %N foO(by difference) 55.19 5.36 7.28 32.17 55.20 5.12 7.15 32.13 Calculated for C9H1004N The free acid showed a decomposition point of 250° C., and corresponds in many respects with 3,4 diox:y phenylalanine (decomposition point 279° C.). The definite identity of the two has not yet been further established, however, since the subst ance has low activity (5 L.U./mg.), and accounts for a negligible fraction of the total activity of the pea diffusate. V. Fractionation with silver acetate: Saturated silver acetate solution waa added to the above water extract until no further precipitate was ing 48 hours in the cold room, (2° C.). formed after stand- ~ne precipitate was filtered off :md decomposed after suspension in water with hydrogen sulfide gas. The silver sulfide formed by this treatment was filtered off, and the resulting water solution concentrated in vacuo. ~ne mother liquor was treated in similar manner, with the excep tion that the acetic acid formed from the silver acetate was removed by ether extraction. Precip itate Total dry weight Leaf uni ts - Mother liauor - - - 4.5 grams 20,000 Enrich.111en t - 45x Total dry weight - - 22.5 gra111s Leaf units 40,000 Enrichment 18X VI. Picric acid fractionation: Excess of a saturat ed water solution of picric acid was added at 2° C. to the water extract obtained from the i nsoluble fraction of step V. A fine yellow precipitate formed which was filtered off, and recrystallized. from water. The p icrate was -47hydrolysed with dilute sulfuric acid, and the picric acid removed by ether extraction. The sulfuric acid was removed as barium sul- fate, and the resulting acetic acid removed by a second ether extraction. The final extract gave no crystalline material but had an activity of 4o,000 L.U./grarn, representing a 400 times enrich- ment. Elementary analysis of the picrate showed no ash, and gave the following composition:* %0 %H %N 1&0 (b;7 difference) Found - - - - - 36.80 2.31 27.62 33.27 Ce,lculated for c11H7N7o8 - - - 36.19 1.99 26.84 35.08 The nitrogen value might i ndicate, that the substance in question is a purine picrate, and its elementary analysis as well as its physical properties correspond closely to those of the hypoxanthine picrate, c11H7N o8 . Adenine and hypoxanthine are the 7 only two purines giving insoluble picrates. The elementary analysis of hyp oxanthine picrate corresponds more closely to the substance in question than does adenine picrate as shovm by the following comparison: Hyp oxanthine Found Adenine carbon "& I 36.2 36.8 36.2 1o1 hydrogen 1.9 2.3 2.2 %nitrogen 26.8 27.6 30.8 %oxygen 35.1 33.3 30.8 Adenine a.n d hy_poxa....-1thine picrates may be distinguished by difference in their physical properties. only in HCl solution. Adenine picrate forms It is highly insoluble in both hot and cold water, decomposes when heated to 279° C. *Mi croanalysis by G. A. Swinehart Hypoxanthine picrate on -48the other hand forms in either neutral or acid solution. insoluble in cold water, more soluble in 200° C. with deco rrrp osi tion. hot. It It is q_uite discolorizes at The pi crate obtained from the pea dif- fusate is formed from neutral solution, very insoluble in cold water, very much more soluble in hot water. It shoved strong discolora tion at 195° C., and decomposed upon heating to higher temperatures. One active substance obtained from the pea diff us ate on these grounds is, therefore, hypoxanthine. Crystalline hypoxanthine wa s f01md active in the leaf test, table 12,with an activity eq_ual to t hat found for the substance obtained by hydrolysis of the picrate (i.e. 40,000 L.U./gra:m). substance The lowest active concentrati on of either is 0.05 mg. per one cc. of 1% sucrose solution. This con- centration represents a dosage of 2.5 r per leaf section, thus having activity comparable to other known growth factors. VII. Fractionatio n of the mother liouor from the silver acetate precinitation: The factors responsible for activity in t his fraction are as yet undetermined. Lead acetate precipitation, charcoal adsorption, HCl-CII oH precipitations, and many other methods have been employed 3 with little enrichment of activity. VIII. Fractionation of the organic acid fraction: The ether soluble fraction, obtained in procedure III, \-.as dried over anhydrous sodium sulfate. The ether was then removed and the residue tal{en up in fresh alconol-free ether. esterified with diaz:omethane ( ffi1 N2 ). 2 The acids were This reagent was prepared from ni troso methyl urea in strongly alkaline \-.ater solution (73), the gas being caught in ether cooled at 5° C. This ether solution of diazomethane was then dried over KOR so that the extract and all -49reagents were dry. Esterification W,. 'l.s carried out at 5° C. by slow addition of the diazomethane to the ether extract of the organic acids. Completion of esterification was indicated by no further evolution of ga s. After esterification was complete, the ether was removed, and the residue transferred to the distilling flask. The type of micro distilling flask used was similar to that used in the isolation of auxin (50). Distilla tion was carried out at a sure of 20 microns, obtained by the use of a 11 pres- high vacuum 11 oil pump. The following fractions were collected from the side arm of the distilling flask: I-60-800 C. II-80-120° C. III-120-180° C. IV-180-210° C. These four fractions were hydrolyzed by r efluxing t wo hours in a saturated alcoholic p ot assium hydroxide solution. The alcohol was removed by vacuum distillation, and the residue taken up in dilute sulfuric acid. tion. The acids were then removed by ether extra c- These acids were tested as the potassium salts, it making no difference in activity whether tested as the sodium or potassium salts as was shown by tests carried out with succinic 2-nd glutaric acids, table 15. Fractions I and II proved to have little activity, the bulk of the activity being found in fractions III, and IV. The residue was inactive. Fraction I 53 mgs. 1.3 L.U./mg. 69 L.U. Fraction II 50 mgs. 2.0 L.U./mg. 100 L.U. Fraction III 48 mgs. 10 L.U./mg. 480 L.U. Fraction IV 50 mgs. 4o L.U./mg. 2,000 L.U. Fr;ictions III and IV give crystalline material from water -50solution under suitable conditions. The structu.re of t he active acid, however, has not yet be en established. The ash of pea diffusat e was shovm to be active in the leaf test with an activity of 6.6 L.U./mg. ~alitative analysis, using a hydrogen sulfide scheme, showed the presence of either potassium or sodium ions, as well as mercuric ion. The presence of mercuric ion is undoubtedly an artifact, being a contaminant from the use of mercuric chloride in the sterilization of the seeds. Potas s ium nitrate was found extremely active in promoting leaf growth, table 16. The effect of potassium nitrate was not, however, additive to that of the crude diffusate. Such an addition was found to increase the activity of the pee, diffusate, but by only 505& of the activity shown by an equivalent concentration of potassium nitrate when tested alone. The following separations were made, in an attempt to establish the contribution of the inorganic salts present in the diffusate to the total activity. Fifteen a,nd three tenths grams of crude diffusate, shown by analysis to contain 12.2% ash, and 15,300 L.tT. were taken up in methyl alcohol to a final concentration of 95d10. It was then placed for 12 hou.rs at -20 O C. The precipitate that had formea_ was centrifuged off, the mother liquor concentrated and again taken up in methyl alcohol to a concentration of nea,rly 1007&. It was again allowed to stand at -20° C. for 12 hours, and the precipitate which formed centrifuged off. for a third time gave no further precipitation. A similar treatment The following dis- tribution of drsJ weight, ash and activity was found: Fraction 1st pp 1 t 2nd pp 't so l uble Total dry in grams 5.3 2.5 7.6 wt. Milligrams of ash L.U. of L.U. of extract ash 960 254 805 10,650 3,560 6.~-00 1,690 5,360 4, 74o -51 ·In the methyl alcohol soluble fr action, t herefore, t he number of leaf units of ash are grea ter t han t he total number of leaf units found in the extract. An acid ether extraction of t his fr2,cti on gave 300 mgs. of extract having a total activity of 2,000 L.U. Assuming that the activity of the ash is diminished by 50% in the presence of the extract, of the 4,74o L.U. present in th~ extract, 2,780 are accounted for by the activity of inorganic salts, and 2,000 by the acti_vi ty of the organic acids, thereby accounting for the total activity found in this fr a ction. The combined precipitates give 8,000 L.U. of ash; and a total of 14,200 L.U. found in the extracts . .Again assuming a 50% reduction in activity of the ash, 4,000 units would be accounted for by the activity of the inorganic salts. Acid ether extraction of these fractions gave no appreciable extract, so that 10,000 L.U. are left une:x:plained. being The purines would be found in this fraction, they very insoluble in alcohol. On the basis of the p revious fractionation, hypoxanthine would account for at least 3,500 of the 10,000 leaf units leaving undetermined 6,500 presumably due to the fraction discussed in procedure VII. It is possible on this basis to make a r ough estimate of the distribution of activity of pea diffusate, which is shown diagramatically in figure 10: Ash 6,800 36.1% Hypoxanthine 3,500 1.8 • 6dl '· Organic acids 2, 000 10.8;& Undet ermined 6,500 34.5% At best t his can be but a rough estimate. The activity of the ash as determined after ashing in air is lar.gely due to and carbonates, 16 . chlorides known to have lm-, activity in the leaf test, table Pea seedlings, and cotyledons are 1mown (19 ) to contain some -52nitrate nitrogen which might incrense t he rel a ti ve activity of the inorganic consituents of the pea diff usate, potassium nitrate being highly active in the leaf test. A portion, at least, of the inorg- anic salts exist as the salts of the organic acids, which would eonsiderably alter their relative activities. Possible synergistic effects betwe en the various active fractions, and effects with possible inactive cofactors would also change the relative activities, though no such effects have been found as yet. bution of activity tive. This distri- must, therefore, be considered as purely tenta- Interactions betwe en the various growth factors will be dis- cussed more fully in the next chapter, together with a discussion of the increase in total number of L.U. during the course of fractionation. This chapter may be summarized as follows: (1) Two different methods emp loyed in the fractionation of pea diff us ate are described. (2) A cryst alline compound of low activity was isolated and its empiric formula established as c H10o4N. 9 It is thought possible that this substance is identical with 3,4 dioxy phenyl alanine. (3) A crystalline picrate was isolated. This picra.te was found upon hydrolysis to give a substance with an activity of 40,000 L.U./gram. This picrate was shown to be identical with the picrate of the purine, hypoxanthine (c11H7N o ). 78 (4) The organic acid fraction from pea diffusate was found to be active. Esterification and high vacuum distillation gave a fraction of high activity distilling between t he temperatures of 180-210° C. at 20 microns pressure. (5) The inorganic salts present in the pea diffus a te were snown to be active. -53(6) A tentative estimate of the distribution of activity in the pea dif'fusate is given as follows: Ash 36.1% H;vpoxanthine 18.6% Organic acids 10. 87~ Undetermined 34.5% -54Table 9* *** General analysis of pea diffusate. Fraction determined Total nitrogen (micro-dumas) Percentage d!Z wt. 4.36 • - · Total nitrogen (micro-Kjeld8.hl) Protein nitrogen o.o Non-protein nitrogen 3.2 Ammonia nitrogen 0.3 Amino nitrogen 1.9 Amide nitrogen 0.05 PolYPeptide nitrogen 0.02 Reducing sugars Sucrose Non-carbohydrate reducing substances Ascorbic acid o.o Ash 12.2 *The author is indebted to Prof. H. ]orsook: and Mr. W. McRary for the nitrogen fraction determinations. Pea diffusate 95 grams 9,000 L.U. ]en~ene and petrol ether extraction 95 gms. 9,000 L.U. Alkaline ether extraction ruble 1 gram 0 L.U. srble 95 gms. 9,000 L.U. 200 mgs. Barium sulfate precipi tatim 0 L.U. 36 gms. 30,000 L,U. 10 gms. 0 L,U. 1 gm. 6,600 1.U. l Acid ether extraction X2.8 o.uble 85 gms. 50,000 L. U. x6 diazomethane distill Mercuric acetate precipi tation ol~ 26 gms. 30,000 L.U. Xll 130-210° C, 20 mm. 4o L.U. /mg. Ice box Inact'ive Silver acetate 26 gms precipitation 30,000 L.U. C9H1004N (5 L.TJ./mg.) 1 olub,le. 4.5 gms. ·r ::9 20,000 L.U. • 22.ry gms. I Picric acid pp 1 t 40,000 L.U. Xl8 C11H N o& (hY.!:oxanthine 77 40,000 L.U./g. picrate) Figure 9. Fractionation of pea diffusate. crude pea diffusate 15.3 grams 12 .2% ash ca. 15 300 L.U. 100% methyl alcohol -20° C. precipitate oluble 7.8 grams - 14,410 L.u. 1.234 grams ash - 3 ,090 L.U. Ash Hypoxantl:iine 4,000 L.U. 3,500 L.U. 7.6 grams - 4,740 L.T.T. 805 mgs. a.sh - 5,360 L.U. Org·a nic a cids Ash .,,,,,. 2,000 LU. 3,,780 L.U. ,/ __,, _,,,,,, __,,. _,,,,. / _,,,,,, / Ash Eyooxanthine / / Organic acids 367i 197;, 11% ~_,,, / •• ,1 Figure 10. Distribution of activity in pea diffusate. Undetermined 34% -55Chapter IX *** Activity of pure substances in leaf grouth. A second method for investigation of the general nature of leaf growth factors has been extensively employed. This method has been the testing of pure compounds of known chemical constitution for activity in the leaf test. The study of such cowpounds has shown that activity in the leaf test is found in four general classes of compounds, (1) amino acids, (2) purines, (3) dicarboxylic acids and (4) inorgenic salts. Table 10 lists a variety of substances found to be active in different biological phenomena which have been tested in the leaf test. In every instance they have been found to be totally inactive with the exceptions of nicotinic acid amide, and ure~ both of which showed slight activity at rather high concentrations. The amino acids that have been tested in the leaf test are list ed in table 11. When radish leaves are used as test objects aspara~in and praline proved the most active. However, in the case of the amino acids it seems likely that the species of plant from which the leaf is taken determines the amino acid requirements; thus when Nicotiana sylvestris is used as the test object arginine proved to be the most active, while with Ravhanus leaves arginine was totally inactive. The growth obtained by the use of amino acids is, hov.rever, never as great as that obtained for the S.S.A. (solution of standard activity), nor are the amino acids active by themselves at high dilution. Yeast nucleic acid was found to possess very definite activity. Upon testing of various crystalline purines adenine proved to be active at the highest dilution (20y per liter) Hypoxanthine had activity at a somewhat higher concen- -56tration, while xanthine, uric acid, guanine and caffeine had sligh t activity, or were tot a.lly ina ctive, table 12. That adenine, hypo- xanthine, or a related purine is the only factor concerned in mesophyll growth seems unlikely not only from the chemical studies of t he pea diffusate already discussecL, but also from t h e fact that in only one or two instances w2.s the arnount of growth obtainable from leaf sections in adenine plus 1% sucrose as great as that obtainable from the S.S.A. This means as shown in table 13, that t he amount of growth at the optimum concentration of adenine was not as great as that obtainable at the optimum concentra tion of the S.S.A. In view of the activity of t he acid ether soluble fraction, various organic acids have be en teste d for a ctivity in leaf growth. The f a tty acids were without activity. The dicarboxylic acids on the other hand have good activity, as shown in table 14. Of the acids tested malonic acid was the lea.st active, the activity of succinic acid being somewhat greater. The acids from glutaric to sebacic had the highest activities, their individual activities being nearly equal. Acids longer t han seb a cic a cid, as decane di- carboxyl ic a cid, showed a decided decrease in activity. Thi s opti- mum activity appears in the dicarboxylic acids from c - to 5 c10 . The amount of growth obtainable fr om leaf sections in acids of the glutaric acid_ group, in contradistinction to adenine, was greater than tha t obta inable from sections gro~m in pea diffusate. It is of interest t hat the presence of a single double bond made no apparent change in a ctivity. Fumaric acicJ_ proved to have the same activity as succinic and decene dicarboxylic a cid to have the same activity as decane di carboxyli c acid. Due to the low pH of the -57solutions, the free acids proved toxic at the higher concentrations. Their salts were, therefore, always used in testing, the potassium salts being generally employed. As shown in table 15, no apparent difference in activity could be found between acids tested as the potassium or as the sodium salts, which should be expected if the acid is active only inside the cell. The pH of the cell sap would then determine the form of acid present within the cell. respect, the In this dicarboxylic acids resemble auxin, which has been shown to be equally active in the Avena test when tested as the free acid, the potassium salt or the sodium salt (D. Bonner 7). The most surprising group of compounds found active in the leaf test is the inorganic salts. ties of those tested. Table 16 gives the activi- Few inorganic salts, of those teste~ have marked activity, these being potassium, sodium, and calcium nitrate, and potassium and sodium chloride. Potassium nitrate is by far the most active, being non-to:xi.c at high concentrations and retaining activity to comparatively low concentrations. lowest active concentration is 0.1 solution. The mg. per cc. of 1% sucrose This corresponds to a concentration of 50 mg. per liter which is high in comparison to the accessory growth factors in general. activities of hormones or On the other hand it corres- ponds to a dosage of 2.5"( per leaf section, which is within hormonal concentration. Concentrations as high as 10 mg. per cc. are without toxicity, while the other active salts tested at similar concentrations were found to be either inhibitory, or toxic. The effects produced by potassium nitrate are often considerable giving increases in area and wet weight of 10076 in 4o hours, i.e. an increase far greater than has been observed for any other active -58substance. clear. What the effect is, or its importance is stil l not Tha t an i mpurity is resp onsib l e for this a ctivity seems u nlikely . Samples obt a ined fr om t hree diff erent manufacturing chemical concerns sh owed. unapp reciable differences in a ctivity, table 17. Whether the effect is produced by the pota ssium-ion, or nitra te ion, or both is not certain since both other potassium and other nitra te s alts have activity. Tha t it i s entirely a permeability phenomenon seems unlikely in t hat calcium nitra te and potassium nitrate are more ne arly additive in their effects t han antagonistic as shown in tabl e 18. Sensitivity of leaf sections to potassium nitrate can be influenced by the previous trea t ment of t he leaves fro m wh ich t he sections are · t aken. Leaves allowed to grow over night in a l o/6 sucrose a..11.0. 1% pota ssium nitra te soluti on were found very much less sensitive to potassium nitra te as well a s nearly insensitive to pea diff usa te when used i n the regula r leaf test, than le aves tha t had been 24 hours in 1% sucrose alone, table 19. The sensitivity to p ea diff usat e a s well as to potassium nitrate, of r adish leaf sections, was found to b'e influenced by t he p revious nutrition of t h e plants from which the leaves were t aken. Leaves of t he same age but co l lected from flats that had been given a ) no nutrient, b) Sh ive 1 s nutrient (see ch apter 11) twice a week , and c) Shive 1 s nutrient every day fro m germina ti on of t he seed were teste d for sensitivity to both pea diffusate and p ot a ssiu.~ nitrate, table 20. Shive 1 s nutrient every day almost abolishes sensitivity in the leaf test. Th is experiment i s of cours e open to t h e ob jection that t he leave s from t he fl a t given nutri ent every day were much larger, and so more mature than t he -59control leaves. The response to pea diffusate might, therefore, naturally be diminished. Possible interactions between these va rious groups of active substances have been investigated. No clear evidence of synergistic effects have been found using the various possible combinations at ma..ri.y different concentrations. concentra tions are as a rule ne a rly The effects at low additive. At higher con- centr2.tions the intera,ction proved inhibitory, i.e. the sum of the individual activities are greater tnan the observed activity in the combination. It was further observed that tne activity of the combination tends to approach that of the compound present at the highest concentration. Whether this means that each group stim- ulates a p a rticular tissue of the mesophy]J, or whether they are all capable of causing growth in a single tissue cannot be settled without further data. It is an interesting correlation, however, that the lamina consists of three main tissues, epidermis, palisade and spongy parenchyma, and that three main classes of active substances are found, purines, organic acids and inorganic salts. During the coD.rse of the fractionation described in chapter VIII the total number of leaf units present in the combined extra cts increased from 9 ,000 to 66,000. During the first two extractions with benzene and petrol ether the total number of units remained constant. The total number of leaf units after the next step, however, increased. In that step inorganic reagents were employed, and possi bly some of the reagents remained in the extract, thereby increasing the total activity. A more likely explanation, however, is that in this step an inhibiting fraction was removed, giving an apparent increase in total activity of the fraction. A s econd -60factor influencing t~is increase, is the separation of the active fractions. As mentioned above, the activity of these substances when tested together is not adc1-itive, but rather the activity of the combination tends toward that of the factor present at the highest concentra tion. As these different active fractions are separated and tested independently the sum of the individual activities will give a total activity higher than that originaHy found. The study of pure substances shows the same general classes of active substances as found by fractionation of the pea diffusate. Fractionation of pea diffusate showed inorganic salts, organic acids, the purine, hypoxanthine, and some as yet undetermined fraction all contributing to the activity. Four general types of compounds were found active by study of pure substances, inorganic salts, dicarboxylic acids, purines, and amino acids. ifont I s 11 phyllocaline 11 would appear,therefore, to be multiple in nature. This chapter may be summarized as follows: (1) A large number of different substances found to be active in different biological phenomena were found inactive in the leaf test. ( 2) The activity of amino acids ws,s found to be species specj_fic. Amino acids were found to have low activities in the leaf test, if active at all. (3) Purines were found active in the leaf test. Adenine was found most active, minimum dosage O.OOlJ,y per leaf section. The maximum growth of leaf sections in adenine was not so great as for pea diffusate. (4) Dicarboxylic acids were fou.'ld active in the leaf test. Highest activity was found for the acids from glutaric (c ) to 5 sebacic (010). The maximum growth of leaf sections in these aci ds - 61was grea ter than for pea diffusate. (5) Inorganic salts were found active in the leaf test. Potassium nitra te \·ms found to be the most active inorganic salt tested. Minimum dosage 2.5ry per leaf section. The maximum growth obt~ined by use of potassium nitrate wa.s far greater ( ca. lOOX) than that obtained from pea diffusate. (6) The interaction between these various active subst ences was found to not be synergistic. were additive. At low concentrations the effects At higher concentra tions the interaction proved inhibitory, the activity of the combination tending to be that of the cor.J_pound present at the highest concentration. (7) The factors involved in the observed increase in total number of leaf -w1its during the course of fractionation of pea diffusate were given as, a) removal of an inhibiting substance, and b) separation of the active fractions. -62Table X *** Substances tested between the concentrations of 0.5 0.005 mg. per cc. of 1~ sucrose solution, and found to be inactive in the leaf test using Ranhanus leaves as test objects. Barbituric acid Pantothenic acid Biotin Vitamin B:i. Estrone Vitamin B2 Indole (3) acetic acid Vitamin B6 lificotinic acid Vi ta.1I1in E Nicotinic acid amide Vitamin K 2-methyl, 6-amino pyrimidine Inositol 2-methyl,6-hydro:xy- pyrimidine Uracil 6-amino pyrimidine Thiazole of vitamin B:i. Urea Table ll *** Activity of amino acids in the leaf test using Raphanus leaves as test objects. Activity on basis of gro\-,th in 1% sucrose solution is equal to 100. Active amino acids Concentration expressed as mg. per cc. of 1% sucrose solution 0.1 0.02 0.5 0.002 Proline 124 114 107 105 Asparagine 116 112 103 101 Threonine 115 110 103 100 d-Valine 112 105 103 100 Glutamic acid Toxic 108 102 100 Alanine 110 100 100 100 Leucine 110 100 100 100 Amino acids tested between the concentrati ons of 0.5-0.005 mg. per one cc. of 1% sucrose solution and found to be inactive: Histidine, Glycine, Aspartic acid Arginine, Citrull ine, Serine, Phenyl-alanine, Cystine, and beta-Alanine. -64Table 12 *** Activities of purines in the leaf test using Raphanus leaves as test objects. Activity on b~sis of growth in 1% sucrose solution eq_ual to 100. Purine tested Concentration e:xpressed as mg. per cc. of l ? sucrose solution 0.1 0.01 0.005 0.05.. 0.5 Adenine 118 117 113 Hypoxanthine 109 116 104 100 Xanthine 115 107 100 100 Caffeine 102 112 108 100 100 Uric acid 118 108 100 100 100 Guanine 100 100 100 100 100 -65Table 13 *** Comparison of the activities of adenine and pea diffusate in the leaf test using ~hanus leaves as test objects, with comparison on several different days. Activity on basis of growth in 17& sucrose solution is equal to 100. Date of test :Act i vity at optimum concen-:Activity at optimum con:tration of pea diffusate :centration of adenine November 5, 193g 125 118 November 9, 1938 135 125 November 12,1938 113 100 November 14,1938 130 110 December 1, 1938 118 107 J anuary 13, 1939 108 -66Table 14 *** Activit±es of dicarboxylic acids in the leaf test using Raphan~ leaves as test objects. 1% sucrose Activity on basis of growth in solution eq~al to 100. Acid tested Malanie .. Succinic: Fu.maric Glutaric Adipic Pimelic Azulaic Sebacic Decene dicarboxylic: Decane dicarboxylic: Concentration expressed as mg. per cc. of 1% sucrose solution 0.2 __0.5 O.Ob 0.02 0.01 110 105 100 115 110 105 100 125 122 116 109 116 108 100 100 104 Table 15 *** Activities of the potassium and sodium salts of dicarboxylic acids in the lea.£ test using ~phanus leaves as test object. Activity on basis of growth in 1% sucrose solution eq_ual to 100. Acid tested Salt Succinic K Na Glutaric K Wa Concentration e:xpressed as mg. per cc. of 1% sucrose solution 1.0 0.1 0.2 114 115 113 113 107 107 120 120 119 110 112 118 Table 16 *** Activities of inorganic salts in the leaf test using Rauhanus leaves a s test object. Act ivity ,on ba sis of growth in 1% sucrose solution equa l to 100 . Inor~a.nic sa.l ts Concentration expressed as mg . per cc. of 1~ sucrose solution 1.0 0.1 2: . 2 5.0 0.05 0.2 KN0 3 175 160 14o 125 110 100 Na!IT03 130 130 120 110 100 100 Ca( No ) 2 3 110 120 110 105 100 100 KCl 130 110 100 NaCl 120 105 100 K2 co 3 124 110 100 Na co 2 3 115 110 100 Ina ctive inorganic salts (J.1Jn4) 2S04, K2S04, Mg( N03) 2 , ( NH4) 2 C03, (liJ114)2N03• Table 17 *** Activity of three different brands of potassium nitrate in the leaf test using Raphanus leaves as test object. Activities on basis of growth in 1% sucrose solution equal 100 . Brand Concentration mg.Jee. 11 Merck 11 11 Kahlbaum 11 "Balcer" 5.0 150 147 149 2.5 140 141 140 1.0 130 132 128 0.3 118 120 118 0.1 106 104 107 0.05 100 100 100 Table 18 *** Interaction of pota.ssiurn nitrate and calcium nitrate in the leaf test, using Ranhanus leaves e~s test object. Activities on basis of growth in 1% sucrose solution equal to 100. Concen- trations expressed in mgs. per cc. of 1% sucrose solution. Calcium nitrate Cone. 1.0 Activity nitrate Activit;y: 124 1.0 135 151 142 132 0.3 120 132 128 124 0.1 108 123 120 110 Potassium Cone. 0.3 0.1 110 100 (Interaction) -70Table 19 *** Sensitivity of leaf sections to potassium nitrate as in- fluenced by previous trea t ment of the leaves from which the secti ons were taken. Raphanus leaves as test object. Activity as growth above sections in 17; sucrose solution equal 100. Concentration of potassium nitrate mg./cc. of 1% sucrose solution Leaves 24 hours in 1% sucrose solution Leaves 24 hours in l ~b sucrose and 1% potassium nitrate solution 155 119 1.0 148 108 0.1 109 100 -71Table 20 *** Sensitivity in the leaf test as influenced by the previous nutrition of the plants from which the leaves were taken. Raphanus leaves as test object. Activity of sections in 1% sucrose expressed as growth above sections in wat er alone equal 100. Activity of sections in pea diffusate and potassium nitrate expressed as growth in 1% sucrose solution e~ual 100 . Sections from leaves of plants having received Shive 1 s nutrient solution : Control Twice Every day :(no nutrient) weekly Treatment Water 100 l ;b sucrose 132 100 100 140 (100) (100 ) (100) l ;i sucrose plus pea diffusate mg. dry weight/cc. 5.0 111 110 102 2.0 112 114 105 0.2 109 108 100 17& sucrose plus potassium nitrate mg.fee. 5.0 142 150 128 1.0 126 118 103 -72Chapter X *** The response of excised pea embry os, and excised immature leaves to known leaf growth factors. Additional assay methods for leaf growth f a ctors were di scus sed in chapter IV. The two methods described were the culture of excised p ea. embryos, and young immature leaves. A few prelimin- ary tests have been made on the resp onse of these two tests to lolo1rm leaf growth factors. Combina tions of different amino acids, either alone or in conjunction with thiamin chloride (fo r good root growth) were found to have little effect upon leaf development in excised pea embry os. Dicarboxylic a cids are also without effect upon leaf growth in excised pea embryos (10). The potassium nitra te concentration in t he medium, however, markedly influenced leaf growth as sh own in table 21. Embryos gro~m in medium containing optimum potassium nitrate coneentration showed increased leaf growth, however, when small supp lements of pea diff usate were added, table 21. This effect of p ea diffusate was found to be largely replaceable by addition s of adenine to t he medium at a concentra tion of 0.1 milligram per liter. Higher concentra tions of adenine were found to be inhibitory, ta.ble 21. Whe t her adenine entirely replaces t he effect of pea diffus a te for p ea embryos has not been definitely p roved, since t h e experiments rep orted here are few, and were not carried for a period of time longer than four weeks. In certain tests, however, and for t his length of t ime, adenine at a suitabl e concentration completely replaced t he effect of pea diffusate. The present data suggest t hat t he beneficj_al eff ect of pea diffusate -73upon leaf growth of excised pea embryos is mainly through the effect upon leaf growth of inorganic salts and adenine. A similar situation was found in the culture of excised etiolated pea seedling leaves. When these were grown upon inorganic salts, sucrose, ancl adenine (o.l mg./1.) the growth was found to be approximately as great as that of leaves gro-wn in a medium containing inorganic salts, sucrose, and pea diffusate. Tne culture of immature first foliage leaves of radish, from plants grown sterilely in the greenhouse, showed that the growth effect of adenine (in conjunction with inorganic salts and sucrose), was as great after a period of five weeks as the growth effect of pea diffusate, table 22. These experiments are again open to criticism in that they are few in number, and that they were maintained only for a period of time (four weeks). short They do, however, indicate tha t the promotive effect of pea diffusa te upon growth of entire excised leaves, as for excised embryos, can be replaced for a period of four weeks by the use of suitable concentrations of potassium nitrate and adenine. The results from these two tests differ from the results of the leaf test in so far as they tend to place much greater emphasis upon the importance of adenine for leaf growth, and might indicate that the factor most strongly limiting leaf growth is a purine, hypoxanthine or adenine. The importance of the purine frac- tion is further substantiated and strengthened by a study of adenine as a possible phytohormone discussed in the next chapter. This section may be summarized as follows: (1) Leaf growth in excised etiolated pea embryos is promoted t hrough t he presence in the medium of potassium nitrate and adenine. -74The optimum concentra tion of adenine wa s found to be not greater than 0.1 milligram per liter. (2) Growth of excised etiolated i mmature pea seedling leaves, as well as of excised i rmnature first foliage l eaves of radish seedlings, is promoted by the p resence of adenine in the medium. (3) In all of these cases the promotive effect of pea diffusate appears to be largely replaceable by adenine and inorganic salts. -75Table 21 *** Lea£ growth of exci s ed pea embryos in response to lea£ growth factors. Concentration Treatment Inorga.~ic medium: potassiwn nitrate mg. /1. 85 (control) Average wet weight per lea£ relative to control 100 114 120 Inorganic medium: (KN"o cone. 850 mg.f,1.) 3 plus pea diffusate mg. dry weight/1. Toxic 144 Inorganic medium: (Irno3 cone. 850 mg./1.) plus adenine 130 0.1 150 -76Table 22 *** Resp onse of isolated immature leaves excised from re.dish seedlings grovm sterilely in the light and cultured 4 weeks. Average wet weight per leaf of t wenty leaves. Weight in mgs. Treatment Wa ter Inorganic s a lt medium plus 1%sucrose 1% Inorganic salt and sucrose medium p lus pea diffus a te mg. dry weig ht/1. 40 Toxic 52.2 20 1% Inorg anic salt and sucrose medium p lus adeni ne mg./1. 0.5 46.6 0.1 52.2 -77Chapter XI The influence of knovm leaf growth factors on the growth of green pl,mts. :Sonner ancl Greene (17,18) have shown that certain plants grown in nutrient sand culture respond with markedly increased growth to small additions of thiamin in the nutrient solution. The effect of such additions was shown to be not only upon the root growth, but upon the growth of the shoots as well. Thiamin has no direct effect upon shoot growth (12,55), but as Went (110) has shown there is a factor present in the r oo ts necessary for the growth of stems. Increased ro ot growth would bring a.bout an in- creased production of this factor, w:licb. in turn would. cause more vigorous sho ot growth. It may well be that a similar case exists in the growth of leaves. It is kno~m tha t older leaves produce a factor required for the growth of irr.mature leaves (37). If their requirements for t hi s factor are not fulfilled, its addition to t he nutrient solution should increase the growth rate of these younger leaves. t hetic area This i n turn would .increase the toti:d photosyn- of the l) lant auxin production. increas ing t he centers of thiamin and The end result should, therefore, be a vigorous growth of the plant as a whole. following experiments more To test t his view the were done. Plants were grovm in washed quartz sa,nd cont ained in 2-gallon glazed crocks SU:pplied with drainage. Tbe crocks were supp lied on alternate days with Shive 1 s R2S5 nu trient solution conta ining the minor elements Fe, Mn, Cu, Zn, Mo, and B. intervening days t he crocks were flushed with water. On t h e Ad<H tions of growth suos tances were made di rectl3r to t he nutrient solution. -78In a few experiments plants were grown in an open garden soil contained in 6 or 8-inch pots. In these cases the growth fa.ctor addi- tions were made to t he water with which the plants were supplied. Photoperiodically sensitive plants were maintained under controlled photoperiod. 11 Long photoperiod 11 below refers to a daily photoperiod of 18-20 hours, the natural dey being supple11 mented by light from Mazda lamps. a daily photoperiod of 9 hours. Short photoperiod 11 refers to In al l the eA'J)eriments described below Cosmos plants were maintained under cond i tions of long photoperiod, while mustard plants were maintained under condi tions of short photoperiod. Dry weight determinations were made at the end of each experiment, that is, unless otherwise noted, approximately 30 days after the beginning of treatment. For these determinations the plants were separated into roots ~nd shoot, sho ot in this case signifying the entire aerial portion of the plant. thiamin In a few cases assays were made upon the dry material (8) by the Phycorn.,v- ces method. That adenine can increase leaf growth of Cosmos is easily seen in figure 11. This figure shows a comparison between control plants and plants treated with adenine at a mg. /1. five weeks after germina tion. concentration of 0.1 In one experiment which is outlined in table 23, seeds of a late flowering variety of Cosmos sulphureus were planted in 20 crocks. When the seedlings were two weeks old they were thinned to 15 per crock , and the crocks were divided into four lots of 5 each. One lot, a control, contiruea to receive standard nutrient solution. The s econd lot was supplied with nutrient solution containing aden i ne at a concentra tion of -790.1 mg. per liter (one part in ten million), the third lot containing adenine 0.01 mg. per liter, and the fourth lot with nutrient solution containing 0.001 mg. of adenine per liter. The plants re- maineQ in the greenhouse under conditions of long photoperiod. After five weeks of treatment the plants were harvested, and. dry weights of roots and shoots determined. Table 23 shows that the increase of shoot dry weight brought about by addition of adenine to the nutrient solution is of a different order of magnitude from the variations in shoot dry weights produced by individual crocks. It is also clear that adenine at concentrations below lry per liter of nutrient solution suffices to increase the production of dry weight of Cosl!l_os plants under the present conditions by approximately threefold. The promotive effect of adenine on the growth of Cosmos has been confirmed in ten separate experiments involving 1300 plants. Similar marked promotive effects of adenine on the growth of mustard(Br~ssica alba) and of cotton have also been observed in sa..~d culture experiments. Adenine has also been found to promote the growth of Cosm~ in soil. Uric acid, which is structural ly related to ad_enine, possesses but slight activity in the leaf test, see table 12. Uric acid, however, is capable of promoting the growth of Cosmos and mustard as is shown diagra.rrrnatically in figure 12. In sand culture experiments uric acid app ears to influence t he growth of leaves very mat erially just as does adenine, and it seems, t herefore, possible that uric aciQ is converted in the intact plant to an active le af growth substance - adenine or hypoxanthine. This view is somewhat strengt hened by the fact that t he growth promoting eff ect brought about by uric acid is of approximately the same -80ma€nitude as that elicited by adenine. The fact that combinations of the two substances give a growth p romoting effe ct app roximately the same as that attributable to either substance alone, table 24, migh t also support the above view were it not for t he f a ct that combinations of thiamin and adenine also are no more p romotive of growth than either substance alone, see below. Growth promoting effects of uric acid have been observed with p ine seedlings (Pinus ramorata) grovm in. soil out of doors. It might be expected that a combinatj_on of the growth promotive effects of thiamin, a root growth factor, and adenine, a leaf gro wth f actor, should. be possible. as is shovm in table 25 Such, however, is not the case, for mustard and in t able 26 for Cosmos. With mustard the plants which received both growth factors actually possessed a somewhat smaller dry weight with either substance alone. than did the plants treated A possible explanation of the non- additive effects of thiamih and adenine may be found in the fact that plants treated with adenine contain much more thiamin than plants not treated with adenine, table 27, and in fact, both Cosmos and mustard when treated with adenine contain nearly as much thiamin as simila r plants treated wi th thiamin (8). investigations of the It is lmown from thiamin contents of diff erent s9 ecies of plants (18) that those species which contain relatively abundant t hiamin (e.g. tomato) are little affectecl in their growth by additions of thiami n. Those sp ecies on the other hand wh:i.ch were found to resp ond to added t hiamin were t hose with relatively small amounts of thiamin. It is possible then that Cosmos or mustard plants treated with adenine produce so much t hiamin that they are not limited in -81their growth by the available amount of t his substance. A simila r case of the interaction of growth factors has been noted by Eonner and Eonner (14) in the case of isolated pea embryos. this case pea embryos supplied with In t hiamin were found to s;y:nth- esize much more ascorbic acid than pea embryos not supplied with thiamin. Although the interaction of growth factors has not been studied in detail it has been found that combinations of thiarnin, nicotinic acid, adenine, estrone; and thiamin, nicotinic acid, adenine, vitamin B6 exert no larger effect on t he growth either of Cosmos or of mustard than does one only of the above substances (8). At the same time that the above adenine and uric acid experiments were carried out similar e1cperiments using the root growth factors, thiamin, nicotinic acid and vitamin B6 wer e carried out (8). It is of interest to note t hat at least in some experi- ments the root growth factors just mentioned influenced the growth of the roots relatively more than that of the sho ots, while adenine and uric acid on the other hand, influenced r oot growth relatively less than did the root growth factors. Thus the mean shoot/root ratio (as derived from dry weights) of the control plants from three representative experiments with Cosmos was approximately 7, while in the same experiments the shoot/root ratio of Cosmos plants treated with leaf growth substances was 10, and t hat of Co sm~ plants treated with root growth substances 4. A similar rela tion obtains for num- erous of the experiments with mustard. Exceptions to this generali- zation, however, were found as the experiment of table 23. That adenine actually functions in these experiments primarily as a leaf growth factor i s indicated b;y the fact that with Coslll~, applications of adenine result in a definite increase in -82leaf area even before a general increase in the vigor of the treated plant has become marked. Figure 13 shows five representative leaves (the youngest full y mature leaf' from each of five separate plants) from three series of plants, talcen two weeks after growth factor application has been started. It may be seen that although the leaves from the thiamin trea ted plants are somewhat larger than those of t he control, those of the adenine treated plants are considerably larger still. That the primary function of aa_enine in these experi- ments is as a leaf gro,ith factor is also indicated by the fact that adenine has no activit3r as a root growth factor (13). The marked promotive effects of the root growth factors thiamin, nicotinic acid, and vitamin ]6, of estrone, and of the leaf growth factors adenine and uric acid on the growth of plants might lead to the impression that the chemical specificity of the growth effects is small. the case (8). Such, however, has been shown to not be Experiments with Cosmos indicate that additions of numerous organic compounds to the nutrient medium are without substantial growth :promoting influence, at least when the substances are applied in concentrations comparable to those in which the plant growth factors exert their influence. Among such substances inactive on the growth of Cosmos, but each active in one or more other physiological processes, were found urea, glutamic acid, and decane dicarboxylic acid (8). Tomato was found not to respond to additions of adenine or uric acid. It is also known that it does not respond to addi- tions of the f actors necessary for the luxuriant growth of isolated tomato roots, thiamin, nicotinic acid, and vitamin BG. Thiamin has been shown to be produced in great abundance by to mato, and it '\tTould seem re a sonable to supp os e, t herefore, t hat t hese other factor s are p ro duced by t he t omato plant in amounts suffi ciently l a rge not to limi t g rowth in the normal p lant. Wheat also failed to respond in sand cul tu.re to any of the above mentioned growth f 2,ctors ( 8 ). It would seem t hat t h ese two much selected crop plants may themselves produce abundant suppl ies of the growth factors at present known. This chapter may be summarized as follows: (1) The growth of Cosmos and mustard plants in sand culture is shown to be promoted by additions of known leaf growth factors, adenine and uric acid. (2) Adenine a nd uric acid appear to :influence primarily leaf size. However, they both exert mark ed effects on t h e general vegetative gro\·J th of p lants. These effects may be s e condary to t h e influence on leaf growth. (3) The effects of thiam i_ n and adenine on g ro11th of Cosmos a..~d nmsta rd i n sand culture are not addi tive. It is suggested that t h is non-addi tive effect is due to t he fact t hat p l ants treated with adenine contain nearly as mu ch t h ia.rnin as p lants treated t h iamin alone. with -84Table 23 *** Effect of adenine on the dry weight acCU11mlation of Cosmos plants. E:,q,eriment started two weeks after planting of seed and harvested after a further f i ve weeks. Fifteen plants per crock. Means of five crocks. Treatment Dry weight of shoots: Dey weight of roots: gms. per crock gms. per crock ( 15 plan_t_s~)_______(_l5"--"p_l_an_t_s'""")_ _ Control (nutrient only) 0.93 ± 0.07 0.106 Adenine 0.1 mg. per liter in nutrient 2.95 ± 0.23 o.l+37 Adenine 0 .01 mg. per lit er in nutrient 2.75 ± 0.15 o.4o4 Adenine 0.001 mg. per liter in nutrient 2.78 + 0.12 0.346 -85Table 24 *** Effects of adenine and uric acid alone and in combination on the growth of Cosmos and mustard plants. Approdmately 45 plants with each trea tment. Treatment started. two weeks after planting and experiment harvested after a further four weeks. Trea tment Total dry weig~t in gms. per plant Cosmos Mustard Control (nutrient alone) o.1 G1 0.079 Adenine 0 .01 mg. /liter 0.226 0 .169 Uric acid 0.01 rng./liter 0.198 0.169 Adenine 0.005 mg./liter + uric acid 0 .005 mg./liter 0 .226 0.161 Table 25 *** Effects of vi t ai-uin :B 1 ancl adenine alone and in combination on the growth of mustard plants. similar results. One of two experiments yielding Treatment started two weeks after planting and plants harvested after a furt her four weeks. Fifty plants per series. Tre atment Total dry weight per p lant; gms. Control (nutrient only) 0.079 Vitamin B::i. 0.01 rng./liter 0.192 Adenine 0.01 rng./liter 0 .169 B~ 0.005 rng./liter + adenine 0.005 mg./1. 0.123 -87Table 26 *** Effects of vitamin B~ and adenine alone and in combination on t he growth of Cosmos plants. Trea tment started two weeks after planting and plants harvested after a further four weeks. plants per series. Fifteen One of three experiments yielding similar results. Gms. dry weight per plant: Root Total Treatment Shoot Control (nutrient only) 0.092 0.0146 0.108 Vi ta.min B .1 0 .01 mg. /liter 0.328 0.103 o.431 Adenine 0.01 mg./liter 0.277 0.0365 0 .301 mg./liter + adenine 0.005 mg./liter 0.361 0.058 o.419 B.1 0.005 Table 27 *** Effect of adenine treatment on the vitamin :Si content of Cosmos and mustard plants. Plants harvested when s i x weeks old, after four weeks of treatment. Treatment Vitamin Bi content of leaves: Cosmos Mustard ~/gm.dry wt:~fplant ~/gm.dry:rfplant wt. Control (nutrient only) 0.121 4.5 Vitamin Bi 0.690 8.4 Adenine o.487 6.6 1.00 Figure 11. Leaves fro m p l ants grown with and wi t hout ade_nine. Control: leaves from Cosmos p lants grown in wa shed sand and watered with Sh ive 1 s nutrient soluti on, five weeks after germination. Adenine: leaves from Cosmos p lants grown under identical condi ti ons but with t he a dd.Hion of 0.1 mg ./1. of adeni ne to t he nutrien t so l ution . COSMOS MUSTftID 400 f- z < _J Cl.. a:: ...., Cl.. v; 200 <.:) ::;; f- ;;: >a:: 0 I CONT. URIC ACID SHOOTS -• I I CONT. uruc ACID ROOTS - CONT. URIC ACID CONT. URIC ACID SHOOTS ROOTS J?igure 12. Effect of uric acid ( 0.01 mg. per liter ) on the growth of Cosmos ancl mustard plants. Experiments harvested ann roximately four weeks after beginning of trea tment. Cosmos = 60 plants per treat ment. Mustard = 45 plants per treatment. Figure 13. Representative le aves from Cosmos plants subjected to various treatments. Youngest mature leaf from each of five :9lants in each series. Leaves te.ken ap:proximately two weeks after beginning of treatment. Control= leaves from plants which received nutrient only. Vitamin Bi= leaves from plan ts which received 0.01 mg. of vitamin Bi per liter of nutrient. Adenine= leaves from plants which received 0.01 mg. of adenine per liter of nutrient. Chapter XII *** Discussion Pea diffusate, lmown to be rich in naturally occurring leaf growth factors, was s e.lected as the crude source best suited for isolation of the factors limiting leaf growth in a normal intact plant. Fractionation of the pea diffusate established several classes of compounds as contributing to the activity of the crude material, one of which was sho"vm to be a purine by the isolation and recognition of hypoxanthine, figure 14. In the course of test- ing other lmown purines for activity in leaf growth, the naturally occurring pur i ne adenine, figure 14, was found to have an activity in the leaf test greater than that of hyi)oxanthine. A naturally occurring pur i ne with activity greater than that of hypoxanthine would seem to cast some doubt upon the as swnp tion that hypoxanthine, isolated from a source rich in naturally occurring leaf growth factors, represents the purine factor limiting leaf growth in a normal intact plant. That hypoxanthine rne.,y occur in relative abundance in the seed and seedlings of severe.l members of the Leguminosae is seen from the report of its isolation (47). isolation of adenine of any legume The on the other hand from the seeds or seedlings ha s not been reported, though it has been reported from t he leaves, seeds, and even pollen of other plants (47). The importance of adenine in many cellular constituents and processes (nucleic acids, and nucleosides) indicates tha t adenine may well occur in the seedlings of Piswn, but in all probability at high dilutions. Its isolation would, therefore, require a concentra- tion greater t han t he four hundrecl ti mes enrichment noted in the -90i s ol a tion of hY11oxantb.ine from pea d.iffusate - see chap ter VIII. Fosse (36) has shown t hat uric a cid me t abol ism in the Leg~i~(!Jae p rocee ds in a manner simila r to that of mammals and_ one might expect, therefore, that adenine metabolism would a lso p roceed along similar lines, the first product of its metabolism be i ng h;rpoxa.11t h ine produced by oxidative d.eamination. That hypoxanthine could arise in part by an in vivo metabolism of ad.en:Lne during t he p repar a tion of t he diffusate, and in part by an in vitro oxidative dearnination of adenine dur .;_ ng the course of fra ctionation p ossible. The conclusion, therefore, seems that the purine factor limit- ing leaf growth in an intact plant is hypoxanthine, may not justified. be The relative activities of the various purines in the J.eaf test wer e found to decrease in the order: adenine, hyp oxanthine , xanthine, and uric aci d with guanine totally inactive. Th is order is the p robable se ~uence of adenine metabolism, i.e., adenine through an oxidative deamination is converted to h;rpoxanthine, which is in turn oxidized to xant:C,.ine. Xanthine is oxidized to uric a cid wh ich :i. s t hen oxidized to allantoin (36). This might indicate that adenine is the active purine, and that the others are active only aft er synthesis into adenine, lowering of the apparent a ctivity of the original purine being , therefore, correlated with the number of step s involved. in this synthesis to adenine. considering adenine A different basis for the limiting purine factor in intact plants is fou...vid from a consideration of t he p ossiol e mechanism involved in t he limita ti on of leaf growth. }for t his purpose t h e role of oth er growth factors shoul d b e consi dered, par ticula rly t he relations~i p of growth f a ctor req_uirement s to cel l ular oxidation systems. No at tempt -91will be made to give a complete list of individua.l organisms requiring a particular factor. Rather a general survey of factors known to limit growth in one or more microo;i:-ganisms or higher plants will be discussed. Cozymase is known to be an acces s ory growth factor for Haemophilus influenzae and allied types (63a). The constituent part of cozymase, nicotinic acid amide, is necessary for the growth of several microorganisms (48). In many cases nicotinic acid is utilized eq_ually well a s in t he growth of pea ro ots (1), or even better as for examp le by the dip t heria bacillus (70). Lactoflavin (vitamin B2 ) requirements are often encountered, as for example in the case of lactic acid bacteria (76). Heme-like requ i rements have also been reported (64,67). Thiamin is required by many different organisms, and in varying stages of degradati on. The intact molecule is required for example by the parasitic fungus Phyto"Ohthora ( gl.~). An organ- ism less fastidious which can use equally well the two halves of the thiamin molecule, t he thiamin pyrimidine and the thiamin t h ia~ole, figure 15, is Phlcomyces blakesleeanus (27,92,95). Pea roots are capable of utilizing even less intact portions (15). The mold Absidia ramosa has been rep orted to require both components for rapid development (94), but can grow slowly in the presence of the pyrimidine alone. Still others develop as r ap idly i n the presence of one comp onent alone as when the whole t hieiain molecule is supplied. This is true of Mucor reJnannianus which needs only t he thia zole constituent (71), and also of the ye as t Rhodotorula (93). A very important fact in regar d to t he requirements for these constituent parts of t he t h iamin molecule is, t hat t hough they app ear -92to be requirements for the particular constituent per se, t hey in reality are an indirect requirement of the organism for thiamin itself. The growth factors enumeratec'L above are ones whose limi t at ion of growth is understandable on the basis of limiting a specific cellular reaction and these factors account for a large proportion of the total number of known growth factor requirements. The re- quirement for factors as biotin (55-57), pantothenic acid (116-119) and ascorbic acid (28,67) are of course well known. These substances in all probability are important in some bio l ogical oxidation (38,75). At the pres ent time, however, their functi on in cellular economy is too obscure to permit of further discussion here. Other chemically well-defined growth factors whose role in cellular economy is even more obscure include auxin (113), cholesterol (28), estrone (55-91), vitamin B6 (68,96), pimelic acid (69), and traumatic acid (16,33,34). Modern developments in the field of cellular metabo l ism have tended to give a unified concept of biological oxidations. Disrega rdi ng the sp ecial processes, there is one mechanism operative t hroughout all metabolism, aerobic or anaerobic, i.e., a shift of metabolic hydrogen (4,75,103). Under aerobic conditions this shift terminates in water, under anaerobic conditions in lactic acid or alcohol. The characterization of metabolism as a hydrogen shift toward oxygen i mp lies that carbon dioxide, the second product of oxidation, does not arise through a direct oxidation of carbon atoms, but rather by catalytic decarboxylation. further Recent developments have emphasized that phosphorylation processes are not limited to the first attack upon hexoses, but that phosphorylation reactions dominate a lmost the entire field of anaerobic reactions (75), thus -93dominating the initial r eactions common to both respir a tion and fermentation. Three main classes of systems nn.ist, t herefore, be concerned, and are foW'l.d in cellular oxidations, i.e. hydrogen transporting systems, decarboxylation systems, and phosphorylation systems. The enzyme systems involved in the initial dehydrogenation of hexoses or their breakdovm products are known to have phosphopyridine nucleotide prosthetic groups, figure 14, (35,104-106). By removal of hydrogen fro m the substrate, this prosthetic group ca.lled codehydrogenase is reduced. It has been shown t hat this rea_uced code- hydrogenase is reoxidized in fermentation by such metabolites as pyruvic acid or acetaldehyde, or in respiration it is reoxidized by diaphorase. Diaphorase, having the probable prosthetic group alloxazine adenine dinucleotide (39,107), figure 15, is in turn oxidized by the cytochrome system which is in turn oxidized via cytochrome oxid.ase by oxygen. As mentioned earlier, a growth factor req_uirement for diphosphopyridine nucleotide (coz~'1Ilase) is kno'l'm. The limitation of growth is then explained (experimental proof for this has been obtained - 63b) on the basis that t he organism in question is W1able to carry out the initial dehydrogenation of the foodstuffs. More common thrua. the requirement for the entire coenzyme, is the requirement for its constituent nicotinic acid amide or its derivative nicotinic acid. Though direct proof is lacking, the requirement for these two factors is interpreted as being actual l y a requirement for t he codehydrogenas e. On this basis the organism is W1able to synthesize nicotinic acid or acid amide, but can, given this one f actor, synthesize the codehydrogenase. The limitation of grO\·,th by these factors is then exp l ained on t he same basis a s the limitation of growth by codehydrogenase deficiency, i.e. inability to carry out the initial dehydrogenation of t he fo odstuffs. Lactoflavin is knovm to be a constituent of the pros- t hetic group of diaphorase (39,107). Thou~h direct proof again is lacking it seems logical to assume that the re quirement for lactoflavin is actually a requirement for diaphorase. is known to be a heme comp ound. Cytochrome Heme-like requirements are, t herefore, understandable on t he grounds of cytochrome requirement, and the aerobic growth requirement of Haemouhilus influenzae for a heme-like substance has been shown by Lwoff ( 64) to probably be a cytochrome requirement. That fewer cases of lactoflavin and heme requirements are found in microorganisms than for nicotinic acid amide might be expected. Although the dehydrogenase is nec- essary for t he initial removal of hydrogen from the fo odstuffs the oxidation of the reduced dehydrogenase may go through other channels, as in many types of fermentation, It might be expected, therefore, that facultative anaerobes are limited by lactofl avin and heme-like substances only una.er aerobic conditions. i nfluenzae is an excellent example. Haemophilus Aerobic growth of this organism requires t he entire codehydrogena se a.no. a heme-like substance. Anaerobic growth on the other hand requires only the codehydrogenase ( 64). Decarboxylations center almost entirely upon the enzyme system carboxylase, the prosthetic group of which has been sho;,m to be thiamin pyrop hosphate (62), figure 15. Abun.dant examp les of thiamin requirements are found, and experimenta l proof has been obtained by Horowitz and Heegaa.rd (46) t hat in pea roots at least, -95thiamin requirements are actual l y re~uirements for cocarboxylase. Growth requirements for thja.min are, therefore, understandable on the basis of metabolism inhibition through lack of a d·e carboxylation system. HY]_)oxanthine has at :present no known function in cellular oxidations. Adenine on t he other hana_ is lmown to be important in biological oxidation systems and could limit growth by a mechm1ism similar to that described for nicotinic acid amide, and thiamin. Adenine could limit metabolism by being an essential con~onent in, 1) a dehydrogenase system anc'4 2) a phosphorylation system. Code- hydrogenase and codiaphorase are both nucleotides, and thus require adenosine phosphate in their synthesis. Organisms are lmown to re- quire either the intact thiamin, thiamin pyrimidine, or thiamin thiazole. Just as these organisms require either thiamin pyri- midine or thiamin thiazole for thiaI.1in, and thiamin for cocarboxylase, leaves might require adenine for codehydrogenase, or codiaphorase. Leaves with ad.enine syn thesis insufficient for codeh_ydro- g enase synthesis would require additional adenine to fulfill their requirements for this f2.ctor, Limit ed adenine synthesis would also affect the third main system involved in biological oxidations mentioned earlier, i.e. phosphorylations. Adenosine phosphates are known to be an important phosphorylation system and are of the utmost importance not only in the initial stages of carbohydrate oxidation, but also in the production of cocarboxylase by phosphorylation of thiamin. At present it is impossible to make any prediction as to which of these mech.:.nisms is operative in leaf growth. An attempt has been made, however, to indicate the possible mechanisms through -96whi ch aclenine c oul d limit growth in a manne r simil a r t o t he me ch an i sm lmovm for othe r groH t h f a ctor s . Thes e mechani s ms do not , of course, rigo rous l y est e,b lish adenine as t he purine f a ctor a ctive in leaf gro,·rth i n t he intact p lant. In vie1:1 of t he f 2,ct tha t ade- nine s hows the h ighest activity i n t h e lea f t e st of any of t he purines te s t ed , and since t he mechan is m of its g rowth limi t a ti on is re adily exp l ainab le on a basis s imilar to t hat of othe r known g rowth f a ctors of the intact p lant, .sdenine would se em to be t h e purine leaf g rowth fi,wtor of the intact plant. Hypoxanth ine would t h en show the same relations h ip to a de nin e as t hiazol e and pyrimidine to t hiami n. The possible ro l e of organi c acids in leaf growth wil l be reconsi de red i n conn ection with t he rol e of i no r gani c s a lt s . It might be well to first consider t neir 2,ctivi t y as measured. in terms of the different te s ts. DicarboxyJ. ic a cids were fo und a ctive i n the leaf test, t he most a ctive 'b e ing acids c ont a ining f ive to te n ca rbon a to:ns. The structure of t he naturally occurring a ct i ve acid has no t yet be en established,, but app e a rs n ot to be one of the common a_ic?.rboxylic acids. In t h e exc is ed pea emb ryo te s t and in the gre en p lant t est , dicarboxylic a cids were fo11i1d ina ctive. In t h e e x cised pea emb r yo and in t he green p l a nt, t h erefore, a f a ctor othe r t han dicarbo:x:ylic a cids app ea r ed to be limiting leaf g rowt h , which wa s s h ow-n to be adenin e in t h e p resence of potassium nitra te. The ro l e of inorgani c salts in leaf g rowth shoul d next be con sidered. Potassium nitrate was found t he most a ctive inorganic salt te s te d . Also a ctive in the leaf t es t were cal cium and so d i um nitrate, t h e activities of calcium nitrate a na_ :p ota ssium nitrate being nearly adc1.it ive. Am·>1onium salts were found tot a lly ina ctive. In the excised p ea embryo t es t p ot as sium nitra t e was foun d to exert a n infl uence up on leaf g rowth, a nd 1:1hi l e n ot demonst r a t ed in the -97present work , it is lmown that t he presence of ni tra.te in the nutrient affects leaf g rowth of green plants gro,,m in sand culture (29 ,41). It has of course been generally observed that an abundance of nitrogen in the soil gives r ank growth of foliage. The striking morph- ological response of certain plants given nitrate has often been reported, thus the most striking result obtained by fertilizing apple trees with nitrate is increased leaf growth (Verner, 100). Similar effects have been noted upon other plants as cotton (45), tobacco (5), and tomato (29). A common subject of investigation for many years has been the relative effects of ammonia and nitrate salts as t he external source of nitrogen upon morphological development. The effects of nitrate and rumnonia on culture of tomato observed by Clark (29) are of pe.rticular interest in con.."lection with t he present work. Clark grew tomatoes in sanct culture, and divided them into two main series; 11 nitrate 11 plants receiving calcium nitra te as ihe sole source of external nitrogen, and 11 ammonium 11 plants receiving ammonium sulfate at the same molar concentration of nitrogen as the nitrate plants. The nitre,te plants showed, after one month, much more luxuriant growth than the ammonium plants; the differences as found by Clark are shown in t able 28, t a~en from h is paper. Total nitrogen in t he leaves of ammonia p lants expressed. as percentage dry weight was higher than in the nitra te plants. Protein nitrogen was very much higher in the leaves of the a.'11Il1oniu.m plants than in the le aves of the nitra te plants, as wel l a s the total soluble nitrogen. Consider ing the total soluble nitrog en, ammonium, amino, asparagine, and glutamine nitrogen were all markedly hi gher in the leaves of the ammonium p lants t han in the leaves of the nitrate plants. Only nitrate and total inorganic nitrogen was higher in -98the nitrate leaves. '.f.1he unknown soluble organic nitrogen was nearly the sc>Jlle in the two series indicating that the increase in total soluble nitrogen in the leaves of the ammonimn 9lants was due mainly to amino and amide nitrogen. One further difference between the two series was found in the concentra tion of organic acids. On the basis of milliequivalents in 100 grams of dry leai' tissue, Clark found 153 in the nitrate plants, 72% of which could be accounted for as oxalic, malic and citric acids. Only 71 were found in the ammonium plants, 8% of which could be accounted for as oxalic, malic and citric acids. The concentration of unknown acids is,therefore, slightly h i gher in the ammonium series. Considering leaf grO\•, th al 0ne, one striking morphological difference between the nitrate and ammonium plants is the greatly increased leaf growth of the nitrate plants. This effect is probably not due directly to amino acid . or amide synthesis. Amino acids and amides, which are known to be active in the leaf test, occurred at higher concentrations in the leaves of the ammonium plants. The effect may be due to increased adenine synthesis, since the increased adenine concentration sufficient to markedly increase leaf growth is far too small to be detected in Clark 1 s gross analyses . possibility, however, are .Against this a) that the adenine precursor may be an amino acid, or tha.t in any case the precursor is probably in the form of reduced nitrogen which would be in e q_ui valent or greater abundance in the ammonium plants; b) that an effect greater than the effect of ni tra.te alone can be obtained with adenine in the excised pea embryo test; c) that the total amount of growth observed in leaf sections floated upon a potassium nitrate solution is many times grea ter t han that obtainable from similar sections floated upon an adenine -99solution. It must be emphasized that these arguments do not rule out the possibility that the increased growth caused by nitrate may be due to increased. adenine synthesis; they do show, however, that other p ossible mechanisms should be considered. Assuming that this effect of nitrate upon leaf growth i s not due primarily to reduced forms of nitrogen, two possibilities only are left, 1) that nitrate itself is directly responsible, and 2) that the effect of ni tra te is due to some secondary reaction during the course of nitra te reduction. HoH· nitra te could act . directly is difficult to under- stand, but the possibility at present exists. As it is kno~m that ma.~y plants are able to accumul a te l arge quantities of nitrate without injury, and that some typical accumulating plants are without exceptionally large leaves, as for example celery (79), the theor-J of direct action seems less likely. duction is still obscure (72). The mechanism of nitrate re- The two important facts that have been established are that nitrate is first reduced to nitrite, and t his reduction can take place in the dark. Whether nitrate reduc- tion takes place under the conditions of the leaf test is not determined. Zaleski (74) showed the probable reduction of nitrate in the dark in detached leaves floated upon sucrose solution. His evidence is inconclusive, however, nor does the work of others (6,101) establish nitrate reduction as occurring under the con- ditions of the leaf test. The catalysis of nitra te reduction in bacteria by a carbon monoxide sensitive iron system (nitrate reductase) has been reported by ~astel (80). vrnet~er this system acts directly upon the nitrate, however, has not been determined, making it difficult to decide whether the nitrate reduction ta.~es place by oxidation of a specific enzyme or by oxidation of a -100- specific metabolite. Nitrate reduction, being strongly ena_othermal, requires an abundance of carbohydrate. Hamner (41) has studied the effect of nitrate adcl.i tion to tomato plants previously depleted of nitrate. He found that in plants with h igh carbohydrate content in the dark, nitrate was detectable in the tops four to six hours after nitra te application, with nitrite appearing about two hours later. Resp iration showed a marked increase subseq~ent to the appearance of nitrate in the tops. He further observed that after fourteen hours the le aves of nitrate treated plan ts were appreciably enlarged. With low carbohydrate reserve, the sa.'Ile phenomenon was found, but considerably delayed. It cannot be determined from his data, however, whether the delayed response in leaf growth was due to a longer time required for the nitrate to arrive in the tops, or whether it was delayed nitrate reduction. As mentioned earlier, Clark (29) found a higher concentration of organic acids present in the nitrate plants than in the ammonium plants. This is of interest in view of the fact that organic acids, as discussed earlier, are knovm to be active in p romoting leaf grovrth. It is, therefore, pos- sible that the action of nitrate i s through the production of a specific acid, this acid being produced during the course of nitrate reduction, indirectly through increased carbohydrate metabolism, or directly t hrough oxidation of a specific precursor. The origin of this acia_ is probably dependent upon wh ich of the possible mechanisms of nitrate re duction discussed above is operative. At the present time, therefore, the role and mechanism of organic acids and inorganic salts in leaf growth must be left in abeyance. In summarizing it can only be said that t her e is the possibility that nitrate 3,s well as organic acids act -101directly in promoting le af growth. There is the second possibility on t he oth er hand , that the nitrate after reduction or during the course of reduction gives rise to secondary substances active in leaf growth. In conclusion, kno°l'm leaf growth f a ctor requirements should be considered in relation to the knovn1 growth factor requirements of the other organs. That the growth factor requirements of leaves are different from those of the other organs is obvious. Leaves require adenine, inorganic salts, and probably an organic acid. Stems require auxin, biotin and caulocaline, wr1ile roots re- q_uire t h iamin, nicotinic acid ancl vi ta.,lin E5. This in itself is not surprising if mutual inter-dependence of the organs is to be found. Considering the point of origin of these various growth f 2,ctors, the leaf is known to be the seat of synthesis for factors controlling t he gro\vth of all other gros s organs in the plant. As shown d:iagrama tic- ally in figure 16, auxin precursor is p roduced in the leaf, from which it is transp orted to the terminal growing bud . Th e growing bud then converts this precursor into active au.."'Cin controlling st em and bud growth in conjunction with secondary factors. Thiamin produced in t he leaf regulate s root growth, and florigen producea. in the leaf controls floral initiation. ing t he ir O\m growt h . p etiole growth. Leaves even produce substances controll- Auxin produced in t he leaf controls ve i n and The factor produced in older le aves affecting the gro wth of younger leaves (Gregory 37) is tentatively assumed to be adenine, thus placing adenine on t he list of known phytohormones. Leaves controlling the gro wth of so many organs might well be expected to have their growth in turn controll ed by at least one other organ, t hus leaving the plant not entirely at the mercy of the cap rices of the leaves. Roots ca..YJ. affect lee.f growth as shown b y -102the increased leaf growth of Cosmos in nutrient sand. culture when given thiamin, Thiamin is totally inactive in le?£ growth, indi- eating that increased leaf growth is a secondary phenomenon correlated with increased root growth. The present work indicates that this factor present in the roots affecting leaf growth is an inorganic salt, nitrate. Nitrate salts are,therefore, tenta- tively suggested as the factor present in the roots and affecting leaf growth. Stem growth, wisely enough, is not controlled solely by the leaf, but must have in addition a factor present in the roots, caulocaline. A rough outline of the system might then be: leaf growth controlled by the roots, and root growth by the leaves, while stem growth is controlled cooperatively b;f roots and leaves. The value of such a system to the plant is seen in the fact that increased root growth gives increased leaf growth, the two of them then together giving increased stem growth and a normal l y proportioned plant. Or increased. leaf growth gives increased root growth and the two again together give i ncreased stem growth, thus preserving the symmetry of the normal l)lant. Reductions in growth rates would act i n the reverse but analogous manner. The original and individual symmetries would not be due to relationsh ips as those just described but might be due to the relative dependence of the various org2J1s upon each other, i.e., upon the autotrophy of the different organs, ultimately determined by the genotype. -103Table 28. *** The wet and. dry weights of tomato p lants receiving only ammoni um sulfate or cal cium nitra te as the external source of nitrogen, from Clark (29). Solution Nitra te Ammonium Dry weight Average fresh Average dry weight exoressed as weight of one %-;f fres h of one plant ;2lant wei~ht :Leaves:Stems :Leaves:Stem; :Roots :Leaves:Stems: gm. gm. gm. gm. gm. % % I 94.4 175.6 10.1 12.5 1.8 10.7 7.1 53.7 4.1 4.1 2.2 10.6 7.6 38.7 Di phosphor-pyridine -dinucleotide OH I H-C-0-P-O- P ~ o-c-H 2 I II - II .I - 2. _ H CJ · o o C-H l 0 OH-C- H rl I H-C-0H 0 OH-r-H 1 0 I 1 H·T-OH H-c_J Lc-H I /N-C-N I I --N H-t 'c-H II 1 •H·C .• •C-C0NH2 H-C~ II IJ . N-C . C-H ~C/ b:I ·~ fi NH1 Adenine Hypoxantine • N-C-NH II JI 2. H-C C- N, I. I C-H • N-C-OH II II . H-C C-N 1 I • 'c-H N=-C-N9 N=C-N~ *(Hypoxanth ine) Figu.re 14 . Alloxazine - adenine-di nucleotide H O H II I H-C- 0-P-O-P-0-C-H I I H-C-OH H-C~ I I H-C-OH H-C-OH I I 0 H-C-OH H-C-OH I I H-C H H-C-H I O II I I j:,~ N, AN'\ CH;( CH;~ ~ c/ c~ r=o t C . N-H '<( 'N~ '8c/ I N-C-N H-~ II ~N-C C-H II t=t H ~H 2. Thiamin-pyrophosphate Figure 15. Auxit1 Precur sor Adenine Thiamin F:i. 57.lre Jnorg. Sa.Lts 16. I n terrelation of various plant 6 r ry,rt;h factors. Jnorg. Salts -104Conclusions *** Detailed su.r;1maries ha ve be en i n cluded at the end of each chapter so t ha t only the most s ignificant facts concerning leaf growth f a ctors need be outlined here. (1) A sati s f a ctory quantita tive bio-ass ay for leaf g rowt h factors is a s fo l lows: Ci rcul a r disk s ca . 19.5 mm2 in area are cut from y oung f i rs t foliage leaves of Ra:phenus. Twenty such d isks are flo a t- ed on the solutions to be tested and allowed to g row on th e se solutions for 30 h ours at 25° C. The tota l wet weigh t of a ll 20 sections from a single solution is t h en determine d by di rect weighing in a stfl..ndard manner. Addi tiona l me t h ods t hat may b e used a re mea surement of leaf growt h in excised embr-.1os, a nd g rowth of e xcised immature le aves. (2) Leaf growth f a ctors a re widely distributed in various natural p roducts and ex tracts. The standa r cl crude extract vii t h which t h e leaf test is calibra ted is a see d diff usate of Pisum s a,tiva. This p ea diff usat e is more active as measured in the lea£ test than any of t he other seed diff usates p r ep ared, or of any of t h e leaf ex tra cts prep a r e d with the exception of a young p alm leaf extract. Urines a re v ery active, cow ur i ne be i ng 100 time s more a ctive t han t he standard p ea diffu s a te prep ara tions. (3) Fracti onati on, and isolati on in part, of t h e active constituen ts of t h e p ea diff usa te sh ows inorganic salts, organ i c a cids, and t he purine hy-1l oxanthine contributing to the a ctivity of t h e crude materia l. Stua_y of pure cry sta,l line sub stances shows t h ree gener al clas s es of substances active a t h igh dilutions p ro moting leaf g rowth, i.e. inorganic salts, organic acia_s, and purines. app ea r, t he r efore, to be mu l t ip le i n natur e . Lea f g rowth f a ctors The f a ctor l i mit j_ng l eaf g rowth in man? diff e rent sp ecies of :p l an ts, h owever, app ear s t o be a -105purine. The purine with h ighe s t leaf promoting activity is adenine, and it is suggested that adenine is the purine f2.ctor li miting leaf growth in normal int a ct plants. (l.~) Adenine in the presence of potassium nitrate largely replaces t he effect of crude pea diffusate in promoting leaf growth in excised pea emb ryos and in immature excised leaves. exerts a Adenine too marked promotive effect upon the vegetative gro wth of p lants in sana_ culture. Since adenine is kno'l'm to be without promotive effect upon root growth , and since the leaves of such adenine treated plants are nmch larger t han the controls, the marked increased general vigor of such plants is thought to be a secondary l)henomenon connected with increased leaf growth. Adenine shoul d ,therefore, be included in t he list of phytohormones. (5) Histological studies indicate that in Ni cotiana taba cum leaves t he nature of the response to p ea diffusate is one of cell enlargement. This enlargement appears most marked in the epidermal an.a_ probably palisade cells, giving rise to p ronounced intercellular air space formation. -106Literature cited. *** 1. A,ddicott, F. and J. Bonner. Nic otinic acid and t he growt h of isolated pea roots. Science 88: 577-578. 1938. 2. Avery, G.S. Structure and development of t he Tobacco leaf. Amer. Jour. :Bot. 20: 565-592. 1933 . 3. -- -----. Differential distr i bution of a phytohormone in the devel oping leaf of Nicotiana , and its relation to polarized growth. Bul l. Torrey Bot. Club. 62: 313-330. 1935. 4. Barron, E.S.G. Cellular oxi dation systems. Phys i ol. Rev. 19: 184-239 . 1939. 5. :Beaumont, .JL:B., C.J. Larsinos, P. Piekenbrock and P.R.Nelson. The as s imilation of nitrogen by tobacco. J. Ag,;. Res.43: 559-567. 1931. 6. Bj8rk ste'n, Johan. 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