Physiology Of The Soybean1

PHYSIOLOGY OF THE SOYBEAN' R. W. Howell United States Regional Soybean Laboratory, Urbana, Illinois

Page I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Germination . . . . . . . . . . . . . . . . . . . ........... A. Environmental Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Metabolism of Germination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Root Growth and Nodulation . . A. Environmental Factors . . . .................. B. Nodulation and Nitrogen Fixation ........................... IV. Top Vegetative Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Environmental Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Metabolism ......... ....................... C. Plant Composition . . . ....................... V. Flowering ...................... ................. .. A. Day Length . . . . . . . . . ....................... B. Chemistry of Flowering .................... ........... C. Other Aspects of Flow ........... D. Varietal Differences in ........... VI. Pod and Seed Development ... ....................... A. Environmental Factors . . . . . . ............. B. Composition of Seed . . . . . . . . ............. VII. Discussion .................................................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction During the rapid expansion of production in recent years there has been coordinated and concentrated research in breeding and genetics, and to a smaller extent in pathology, of the soybean [Glycine nuzx (L.) Merr.]. There has been relatively much less work in physiology, except in specialized fields such as photoperiodism and nitrogen fixation, and practically no coordination or evaluation of work on soybean physiology in terms of production problems. Certain aspects of soybean physiology were included in the reviews of Williams (1950) and Weiss (1949), but developments of the past decade 1

Publication No. 338 of the U. S. Regional Soybean Laboratory, Urbana, Illinois. 265

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as well as the wide scopes of those reviews make a specific review of soybean physiology timely now. II. Germination

The physiology of seed germination was reviewed by Toole et al. (1956) and Crocker and Barton (1953). Weiss (1949) reviewed factors affecting viability of soybean seed. As defined in the dictionary (Friend and Guralnik, 1958), germination might be regarded as the resumption of growth of the embryo in the seed. A more practical and useful definition is: “The emergence and development from the seed embryo of those essential structures which . . . are indicative of the ability to produce a normal plant under favorable conditions” (U. S. Department of Agriculture, 1952). This definition is used here. Soybean seeds lack a specific light requirement for germination, but they are no less dependent than other seeds on suitable conditions oE moisture, temperature, and aeration. Also required are suitable internal conditions-available food material, metabolic systems for using it, proper balances of growth regulators, and a structure that facilitates the transport required for life and growth. Germination involves mobilization and utilization of food and energy reserves, in contrast to seed development, in which there is a net accumulation of energy materials. In the field, soybean seedlings begin to emerge within 5 to 7 days, depending on depth of planting, soil moisture, and temperature. Depth of planting is important mainly because of moisture relations, greater depth being required in dry soil.

A. ENVIRONMENTAL FACTORS Hunter and Erickson (1952) found that a moisture content of about 50 per cent was required for germination of soybean seed. Moisture required for germination of corn was about 30 per cent, rice 26 per cent, and sugar beets 31 per cent. Expressed in terms of soil-moisture tension, corn germinated at a tension of 12.5 atm. whereas soybeans failed to germinate if the tension exceeded 6.6 atm. Excessive moisture is unfavorable for germination. Water-soaked seeds show reduced germination (Yamamoto, 1955), and in sand germination tests a sand-moisture content of over 15 per cent results in lower germination percentages ( Delouche, 1953). Seeds can be allowed to imbibe up to twice their weight in water, and if they are dried quickly in a stream of air their germination is reduced only slightly. Although oxygen has been shown to be of significance in certain cases

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of seed dormancy (Crocker and Barton, 1953) and is considered generally necessary for germination (Toole et al., 1956), there is little information concerning quantitative effects of oxygen level on germination. Excess water probably interfers with oxygen supply (U. S . Department of Agriculture, 1952; Ohmura and Howell, 1960). Temperature effects on germination have been studied extensively. Delouche (1953) reported maximum germination in the shortest time with a constant temperature of 86" F. It took about twice as long to reach a given percentage of germination at 68". There was no evidence of need for an alternating temperature. Inouye (1953), using Japanese varieties, reports an optimum germination temperature of 93" to 97", a minimum of 36" to 39", and maximum of 108" to 111". Various methods of testing germination and of interpreting test results are described in Agricultural Handbook 30 (U. S. Department of Agriculture, 1952). Results of laboratory germination tests in quartz sand by the method of the Association of Official Seed Analysts were closely correlated with field germination (Sherf, 1953). Germination in builder's sand is equally good when care is taken to use uncontaminated sand ( R . L. Bernard, personal communication) . A rapid chemical method of testing viability would be very useful. The most common test of this sort uses the staining of embryos by triphenyltetrazolium chloride as an index of viability. This test, proposed by Lakon ( 1942), was first used on soybeans by Porter et al. (1947). The staining of the interior tissues of the plumule seems to be the best index of viability. The test is therefore hard to use on soybeans because of the difficulty of accurately splitting the plumule. Moisture content of immature soybean seeds exceeds the 50 per cent level required for germination (Hunter and Erickson, 1952) until shortly before maturity, yet germination of a seed in the pod is rare. This is probably not due to restriction of oxygen movement by the pod, since oxygen uptake by intact ripening pods and seeds is practically the same as that of seeds and pods after shelling (Howell et al., 1959). Ozaki et al. (1956) found that seeds harvested 35 days after flowering would germinate if allowed to "after-ripen" in air for 10 to 20 days. At the U. S. Regional Soybean Laboratory, we have found that seeds harvested at the 75-mg. stage (about half the final dry weight) failed to germinate immediately after harvest or if shelled and allowed to afterripen at room temperature until constant fresh weight was attained. However, if the seeds were allowed to after-ripen in the pod at room temperature until constant fresh weight was attained (about 1 week) or if they were soaked 2 to 3 hours in deionized water, a high germination percentage was obtained ( Galitz, 1958).

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An extract has been obtained from immature seeds which inhibits germination of radicles excised from mature seeds (Galitz and Howell, 1959). A substance which inhibits germination and which absorbs at 260 mp has been isolated from this extract on a Dowex-1 column. The inhibitor is an uncoupler of oxidative phosphorylation and appears to be identical with a substance that is synthesized in soybean seedlings sprayed with 2,4-dichlorophenoxyacetic acid (2,4-D ) (Key and Galitz, 1959). During after-ripening this inhibitor is presumably metabolized to a noninhibitory form.

B. METABOLISM OF GERMINATION Respiratory activity during germination of soybeans is characterized by increased oxygen consumption and by changes in substrate specificity that may be related to changes in the pattern of utilizing food reserves. The tricarboxylic acid cycle is present and, as in other tissues, a high level of oxidative activity is concentrated in the mitochondria (Funahashi et al., 1953; Switzer and Smith, 1957; Howell, 1958). Akazawa et al. ( 1953) have reported transamination and amino acid synthesis by cytoplasmic particles (presumably mitochondria) from soybean seedlings. Both the amount of mitochondria1 nitrogen and the uptake of oxygen per unit of nitrogen reach a peak about 5 days after the start of germination (Howell, 1958). Also by the fifth day the “glyoxylate shunt” (Kornberg and Beevers, 1957), by which fatty acids can be converted to carbohydrates, appears to be active (Howell, 1958; Carpenter and Beevers, 1958, 1959) . Although the mitochondria are generally regarded as accounting for most of the oxygen uptake in tissues, recent experiments (T. Ohmura, unpublished) have indicated that an oxidative system in the soluble fraction of the tissue, when provided with cofactors contained in the native fat, may account for as much respiration as do the mitochondria. Holman (1948) found that the oil content of cotyledons decreased during germination and after 15 days was only about 2 per cent. The iodine number of the oil decreased from about 140 to 120. Lipoxidase was at a maximum on the fourth day, but fell very rapidly by the ninth day to 10 per cent of its maximum. Catalase activity showed a sharp peak on the fifth day. These enzymes use peroxides as substrates and are of interest in fat metabolism because of the probable formation of peroxides during the breakdown of unsaturated fatty acids (Stumpf and Bradbeer, 1959). Kahn (1959) reported an accelerated loss of oil following a peak in Iipase activity which was reached on the fifth day. Kahn et al. (1958) showed that, in situ, the fat fraction of germinating soybean cotyledons occurs in droplets surrounded by a membrane probably containing pro-

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tein, lipid phosphate, nucleic acid, and water but no carbohydrate. During germination the proportion of nontriglyceride components in the “native fat” fraction decreases linearly with time. Although the details of fatty acid degradation are not yet clear, the mitochondria of germinating soybean cotyledons are implicated as sites of at least some of the reactions of fatty acid degradation. They contain significant levels of lipoxidase, peroxidase, and catalase, they can oxidize linolenic acid (Kahn, 1959), and they may contain the enzymes of the “glyoxylate shunt” (Howell, 1958). McAlister and Krober (1951) showed that carbohydrates are depleted much more rapidly than the fat fraction, being down practically to zero by the third day. Protein decreased at about the same rate as oil for the first 2 weeks, but much more slowly thereafter. Five weeks after planting, each cotyledon contained about 2 mg. of protein. The importance of changes in vitamins, amino acids, etc., to the germinating plant in most cases is poorly understood, but substances in these categories are of particular interest in animal nutrition. McKinney et al. (1958) found that ascorbic acid, which was absent in mature beans, appeared soon after the start of germination and increased rapidly during the first 4 days. Sugimoto (1954), in a 10-day test comparing germination in the light and in the dark, found the highest ascorbic acid level on the seventh day in the light and on the tenth in the dark. Ascorbic acid in the reduced form in germinating soybeans was 0.178 mg. per gram and in the oxidized form 0.086 in a study by Sugawara (1953). Comparable figures for corn were 0.045 and 0.068 mg. per gram. Mee (1949), Sugimoto ( 1954), and McKinney et al. (1958) all found total thiamin (vitamin B,) essentially constant (12 to 14 pg. per gram according to McKinney et al.) during germination. But Mee found that “combined” thiamin ( i.e., thiamin pyrophosphate or cocarboxylase) increased initially at the start of germination. He suggested that “early in growth the metabolism of the seed must require phosphorylation of thiamin, but this process is completely reversed in about 4 days.” By this time the seed’s supply of carbohydrate is exhausted, so that there may be no further need for the system decarboxylating pyruvate. Sugimoto found that changes in thiamin, as well as in vitamin Bz (riboflavin), cystine, cysteine, and methionine, were more rapid in the light than in the dark. Richardson and Axelrod (1957) observed a decrease in inositol during germination of soybeans and considered such a decrease to be general among higher plants, although Burkholder and McVeigh (1945) reported an initial increase in inositol in germinating soybeans and several other

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crops. Inositol may have significance in the formation of nodules (Raggio et al., 1959). 111. Root Growth and Nodulation The health and vigor of the entire plant are conditioned by the distribution and function of the roots. A continuous supply of water and nutrients is an obvious requirement, Less apparent but perhaps no less important is the control of top growth by hormones formed in the roots (Went, 1938; Howell and Skoog, 1955). A special requirement in the case of legumes is the development of the symbiotic nitrogen-fixing system which makes the plant autotrophic with respect to this element. It follows, therefore, that knowledge of root growth patterns and of environmental effects is important to an understanding of total plant performance. The soybean root system is characterized by a primary root, which may penetrate as far as 5 feet (Borst and Thatcher, 1931). Under conditions unfavorable for deep penetration, the taproot is less important and the branch roots more so. Under any conditions, most of the root system, according to Borst and Thatcher, is usually in the upper 2 feet of soil. As the plant develops, root growth continues until about the time seed development begins, after which total root weight decreases. Even before the cessation of root growth, above-ground organs grow more rapidly than the root system, so that the top:root ratio increases steadily throughout the life of the plant, Top:root ratio has little significance in itself since it is altered by developmental and environmental conditions (Roberts and Struckmeyer, 1946). The zonation, organization, and growth of the primary root of the soybean were described recently by Sun (1955b, 1957a). The root tip consists of three regions of increasing maturity: the promeristem, the primary meristem, and the primary permanent tissues. The promeristem consists of eight to fourteen tiers of cells and extends about 100 p in length. The primary meristem is about 200 p in length. Of the primary permanent tissues, the most conspicuous is the primary xylem, which is in the form of a tetrarch. Lateral roots arise in the pericycle at loci directly opposite the ridges of the tetrarch xylem and develop acropetally in four longitudinal rows beginning just below the hypocotyl. The epidermis is uniseriate and consists of closely packed elongated cells with thin walls. Formation of root hairs began on the fourth day after planting in Sun’s study. A. ENVIRONMENTAL FACTORS It is axiomatic that root growth and activity are influenced by environmental variables. Temperature, moisture conditions, aeration, and

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supply of nutrients are factors of obvious importance. The first three of these will be discussed in this section, but, except for incidental reference, the role of nutrient elements is discussed in the accompanying paper by Ohlrogge. 1 . Temperature Earley and Cartter (1945) found that, under greenhouse gravel-culture conditions, temperature variations between 54" and 99" F. had only a slight effect on root growth (dry weight). But although differences were small and barely reached the 5 per cent level of statistical significance, the greatest weights of roots in four experiments, which included temperatures from 45" to 99", were obtained at either 81" or

90". Both the total and the distribution of cation uptake varies with temperature. Wallace (1957) found that potassium uptake by soybean roots increased over the temperature range from 54" to 90" F., whereas calcium and magnesium uptake decreased by more than an equivalent amount. Kahn and Hanson (1957) found that a 4" increase in temperature (77" versus 81") increased the velocity constant (V,) for potassium accumulation from 1.18 to 1.30 microequivalents per hour per gram fresh weight, but they observed no effect of temperature on the equilibrium constant for potassium accumulation in either soybeans or corn.

2. Moisture Growth of roots as well as of the rest of the plant is affected by soilmoisture conditions. Soybeans use water from progressively greater depths throughout the season. Swan (1959) found water use directly in the row to be double that at points one-quarter and one-half the distance between rows. However, when moisture supply was increased by irrigation, the water use from the between-row locations increased to about two-thirds that in the row. A water table in the root zone is injurious (Fukui et al., 1954), especially if it is near the surface early in the season and later falls. Flooded soybean plants may develop aerenchyma tissues, which serve to aerate the underground structures and increase the tolerance to flooding (Fukui, 1956; Arikado, 1954). The wild soybean, (Glycine ussuriensis Regel and Maack) developed a general aerenchyma in aerial and root tissues, a vigorous adventitious root system and yielded as well when flooded continuously from July 17 to maturity (planting date: July 1 ) as when soil moisture was maintained at 50 per cent of "water capacity." Plants of a cultivated variety developed only a lenticel-like aerenchyma and failed to survive flooding during July 17 to 31 or August 1 to 31.

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3. Aeration The importance of an adequate oxygen supply to the general metabolic activities of the root was stressed by Hoagland and Broyer (1936) and is Srmly established by the results of many workers. According to the “carrier” hypothesis of ion uptake, as discussed by Epstein (1956), ions may diffuse into certain “outer” spaces of cells, the concentration there approaching equilibrium with that of the external medium. Ions in “outer” spaces can be easily removed by washing or exchange. Transport to “inner” spaces, however, is postulated to involve a “carrier” or “pump” system and in any event is dependent on respiratory energy (Hoagland, 1948; Russell, 1952; Epstein, 1956). “Inner” spaces may be vacuoles, cytoplasm, mitochondria, or other sites to which ions are irreversibly transported ( Laties, 1959). Most of the normal respiration of soybean roots can be accounted for in the mitochondria, which contain a conventional Krebs cycle type of metabolic system (Key et al., 1960) and thus require a supply of oxygen. Soybean roots respond to changes in oxygen tension. Using solution culture techniques, Shive (1941) and Gilbert and Shive (1942) found that soybeans grew best when an oxygen concentration of about 6 p.p.m. was maintained in the solution. In the absence of aeration, the roots reduced the oxygen content of the solution to practically zero in 6 to 9 hours. With no dissolved oxygen in the nutrient solution, root growth was about half that with optimum oxygen. Sun (195%) found that the length of primary roots and the number of secondary roots after 3 weeks’ growth in aerated solutions were each about three times the comparable values in nonaerated solutions. Root hairs, on the other hand, were abundant in nonaerated solutions, but sparse if aerated. Hopkins et al. (1950), as a result of studies in which gas mixtures containing 0.5 to 21 per cent oxygen were supplied to the roots of several species growing in 8-mesh quartz sand, concluded that soybeans are able to maintain growth processes at oxygen levels as low as 1.5 per cent. Only when the oxygen supply to the roots was reduced below this level was top growth significantly restricted. The rates of potassium, calcium, and phosphorus uptake under 0.5 per cent oxygen conditions were 25, 16, and 45 per cent less than the rates in air. Magnesium and the minor elements were unaffected and sodium was increased 67 per cent by the reduced oxygen level. The production of respiratory oxygen by reduction of NO3 was suggested by Arnon (1937) and may be of general significance. Evidence from the work of Shive (1941) and Gilbert and Shive (1945) indicates

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that NOs may serve as an oxygen donor in soybean roots. Possibly the ability of soybeans to withstand low oxygen tensions in the root environment was at least partly due to availability of NO3 in the nutrient solution. 4. Herbicides Although most attention in herbicide studies has been given to effects on plant tops, treatment of soybeans with 2,4-dichlorophenoxyaceticacid (2,4-D) also injures and may ultimately kill the roots. The orderly development of primary tissues, particularly within the stele and inner cortex, is disrupted by 2,4-D treatment (Sun, 1956). In these regions cells are stimulated to a high state of meristematic activity, but the resulting mass of cells is nonpolarized and lacks the lateral root primordia that are characteristic of normal roots. The hypocotyl is similarly disorganized. The pith cells become meristematic and the pith develops abnormal vascular strands. In addition to inhibiting elongation, 2,4-D inhibits straightening of the hypocotyl hook when applied to seedlings (Key et al., 1960). Oxygen uptake was decreased about 50 per cent in the apical section and about 20 per cent in the older section of the root. Williams (1953) tested 93 varieties in solutions containing 0.5, 1.0, or 2.5 p.p.m. 2,4-D. At the lowest level, 4 varieties had root growth of 80 per cent or more of the control while 5 varieties had 50 per cent or less. Inhibition was increased by increasing the 2,4-D level. At all levels there was a wide range in varietal responses, but there was no variety which was uninhibited at even the lowest level. Injury to roots was greatly aggravated by high nitrogen levels (Wolf et al., 1950). B. NODWLATION AND NITROGEN FIXATION 1. Growth Factors Raggio and Raggio (1956) have shown that, in order to nodulate, soybean seedlings require a factor from the cotyledons. The factor is mobilized very rapidly, because nodules were formed on about 50 per cent of 2O-mm.-long primary roots excised from 4-day-old seedlings and cultured on a sucrose-agar medium (Raggio et al., 1957). Of many vitamins, amino acids, growth hormones, nucleotides, and extracts tested, only inositol enhanced nodulation. Both the percentage of roots nodulating and the number of nodules per root were increased by the addition of meso-inositol (Raggio et aZ., 1959).

2. Relation of Age of Plant t o Nitrogen Fixation The speed with which nodules are formed and become active in nitrogen fixation on young plants is impressive. Bergersen (1958) observed that the first nodules appeared on LINCOLN soybeans 9 days after

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planting and nitrogen fixation began about 2 weeks later. Even so, inoculated soybean plants may lack sufficient nitrogen after seed reserves are largely mobilized-about 2 weeks after planting ( McAlister and Krober, 1951)-and before the symbiotic system becomes fully active (Fred et al., 1938). In the study of Bergersen the seedlings became increasingly yellow until about 15 days after the start of nodulation, when they “became green overnight.” There is lack of agreement as to how long active nitrogen fixation continues. Using winter-grown plants which flowered in “2 to 3 weeks after germination,” Bergersen found that total plant nitrogen increased at essentially a constant rate from about 2 weeks after the first nodules appeared until about 4 weeks later, when nodule decay started. By 58 days after planting, or 7 weeks after the first nodule was formed, nitrogen fixation had ceased. At this time the plants ‘‘bore 4 trifoliolate leaves and young pods with seeds about 2 mm. in diameter” (F. J. Bergersen, personal communication ). Summer-grown plants produced successive “crops” of nodules at intervals of about 14 days. Aprison et al. (1954) report substantial fixation by nodules excised from HAWKEYE soybeans 80 days after planting. At Madison, Wisconsin, where these experiments were conducted, HAWKEYE matures in about 135 days. It can therefore be assumed that these plants were in a late flowering stage at the time of harvest. Magee and Burris (1954) observed that nitrogen fixation by excised nodules “increased roughly with increasing size of the nodules from young, actively growing plants” but declined as the growth rate slowed with blooming. Bond (1936), on the other hand, reported the highest daily rate of nitrogen fixation per plant during the period of pod set and development. Total weight of nodules increased throughout the life of the plant, and just before maturity totaled 2.5 g. per eight plants in pot culture. Thus the data pertaining to the duration of nitrogen fixation are conflicting. Wilson (1940, p. 155) refers to the harvest of pea plants “when the pods had set and nitrogen fixation had probably ceased,” but perhaps the implication that fixation does not occur during pod and seed development is not applicable to soybeans. There is clearly a requirement for substantial amounts of nitrogen during the period of pod and seed development (Hammond et al., 1951; Togari et al., 1955), but soybeans grown in nutrient solution fail to accumulate enough nitrogen up to mid-bloom to s&ce for subsequent growth (Lathwell and Evans, 1951). If fixation ceases by the start of seed development a considerable amount of nitrogen must be supplied from the soil. Aprison and Burris (1952) were the first to report nitrogen-fixation by excised nodules. The most active nodules are 5 to 6 mm. in diameter

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(Magee and Burris, 1954; Aprison et al., 1954). There is a sharp temperature optimum at 77” F. (Aprison et d.,1954). Excised nodules rapidly lose the ability to fix nitrogen, the amount fixed in 6 hours being less than twice that fixed in 1 hour. Fixation takes place in water and is not enhanced by addition of a number of common respiratory substrate:; and cofactors. Nitrogen-fixation by free-living cultures of Rhizabiurn has not been established although there have been unconfirmed claims. Bergersen and Briggs (1958) observed that in the nodules up to six “bacteroids” are grouped within double membranes. Bergersen and Wilson (1959) concluded that N-fixation occurs not in the bacteroids, but in the membrane envelope which surrounds them.

3. Genetic and Environmental Factors It is well known that there are genetic differences in ability of soybean lines to nodulate (Weiss, 1949, p. 106). Williams and Lynch ( 1954) have found a nonnodulation response that is determined by a single gene pair. Failure to nodulate is not due to absence of bacteria from nonnodulating plants (Clark, 1957). Clark was unable to transfer the ability to nodulate by approach-grafts below the first trifolioliate leaf when plants were 3 to 4 weeks old but this could have been due to lack of the cotyledon factor of Raggio and Raggio (1956). The importance of soil factors in nodulation was also indicated by Clark‘s work. He found bacterial strains which could produce nodules on the “nonnodulating” line in sand culture, although not in any of three soils that were used. Whether these nodules fixed nitrogen was not reported. Strains of Rhizobium that induce chlorosis have been found, although soybean varieties are not equally susceptible (Johnson et al., 1958; Erdman et al., 1957). The chlorosis-inducing factor is extractable in water and retains its activity in solution (Johnson et al., 1959). Nodule development is influenced by processes in the leaf (Sironval et al., 1957; Bonnier and Sironval, 1956). More and heavier, as well as more effective, nodules were produced under long days than short. Sironval (1958) stated that nodule formation appears to be parallel and related to chlorophyll formation. Bach et aZ. (1958) showed the dependence of nitrogen fixation on photosynthesis or, in the case of sliced nodules, an external supply of sugar, and Kamata (1957) showed that nodulation is promoted by foliar application of sugars. Bach et al. also reported relatively high C14 activity in an unidentified component of the organic acid extract of nodules from plants receiving C1402 in the light. This substance may have significance in N-fixation.

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4. Metabolism of Nitrogen Fixation

Active soybean nodules contain a characteristic red porphyrin hemoprotein (Ishizawa and Toyoda, 1955). It is assumed that this hemoprotein is required for nitrogen fixation, since nodules in which the pigment has turned green fail to reduce hydroxylamine to ammonia as do those with red color (Virtanen et al., 1954). Hamilton et al. (1957) showed that this protein is oxidized by Nz and that the oxidized form is reduced by Hz, so that it could act as an oxidation-reduction catalyst or hydrogen carrier in nitrogen fixation. Little (1949), Richmond et al. (1954), and Richmond and Salomon (1955) have shown that the red pigment from soybean nodules is very similar to, but is not identical with, hemoglobin from animals. Thorogood (1957) found that three and possibly four protoporphyrin proteins can be isolated from soybean nodules. Hunt (1951) found five ninhydrin-staining substances that seemed to be unique to nodules and suggested that these compounds might be of significance in nitrogen assimilation. Nodules have a very active respiratory system, as must be expected since the fixation of nitrogen by reduction of Nz or, as recently suggested by Mozen and Burris (1954), of NzO,involves a substantial increase in the energy level of the nitrogen atoms (Lange, 1949). Practically no evidence of nitrogen fixation was obtained under anaerobic conditions (Hoch et aZ., 1957), and maximum fixation required an oxygen partial pressure of half an atmosphere (Burris et al., 1955). Evans (1954) and Cheniae and Evans (1957) reported data showing that nitrate reductase is concerned in N-fixation. This enzyme requires molybdenum. Succinate appears to be the hydrogen donor for nodule nitrate reductase, thus providing a metabolic basis for the dependence of fixation on aerobic respiration. Bergersen ( 1958), however, found very incomplete oxidation of Krebs cycle intermediates by either rhizobia from pure cultures or bacteroids from nodules, and suggested that this cycle does not operate in these bacteria. Asprey and Bond (1941) found oxygen uptake by nodules to be two to three times that by soybean roots. They obtained respiratory quotients (R.Q.) up to 1.25 for nodules, possibly indicating some anaerobic respiration, and found no difference in R.Q. between large and small nodules. Thus they concluded that an adequate oxygen supply exists throughout the nodules. Allison et aZ. (1940a,b ) , on the other hand, found R.Q. values in nodules as high as 2.0, the larger values being obtained with large nodules.

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5. Other Effects of Nodulur Activity Is nodular activity beneficial to the plant in ways other than supplying nitrogen? Hawkes (1952) obtained maximum yields with LINCOLN variety only when the plants were well nodulated, even though high nitrogen levels were supplied to uninoculated plots. Maximum yields could not be obtained from nodulation alone, however, added nitrogen being required. Nodulation seemed to facilitate phosphorus accumulation, in contrast to the decrease in phosphorus that resulted from an increase in external nitrogen supply. Chesnin and Haghiri (1957), on the other hand, found no yield differences between nodulating and nonnodulating lines in nutrient solutions. Nodulation reduced the potassium percentage in the roots (Haghiri and Chesnin, 1957) but did not affect calcium, magnesium, and phosphorus levels. C. R. Weber (personal communication) compared the responses of two lines, which were isogenic except for the ability to nodulate, to added nitrogen on both high productivity and low productivity soils. On the high-productivity soil, differences were small. On the other soil, productivity of which was lowered by plowing in 20 tons of corn cobs, yield of the nonnodulater was increased to the level of the nodulator by addition of 600 pounds nitrogen per acre. Lynch and Sears (1952) found no effect of inoculation on yields on eight soil experiment fields, although other treatments such as manure and lime gave yield increases.

IV. Top Vegetative Growth Under favorable conditions of temperature and moisture the soybean seedling emerges 4 to 7 days after planting. Nodes in the main axis are rapidly differentiated, as many as twenty being detectable macroscopically or microscopically by the time the fifth compound leaf has expanded (H. A. Borthwick, personal communication). Initiation of floral primordia begins within about 3 weeks and flowering within 6 to 8 weeks when varieties are grown within their areas of adaptation. Pods are normally visible about 10 days to 2 weeks after the start of flowering. Except at the cotyledonary and second nodes, the soybean has a single trifoliolate leaf at each node. The opposite unifoliolate leaves of the second node and the first trifoliolate leaf are preformed in the plumule (Williams, 1950). Either a vegetative or a floral bud may be produced in the axils of most leaves. Plants may appear determinate or indeterminate in growth habit. This is influenced by environmenta1 conditions, although some varieties, such as LEE and WRMAN, have a definite terminal raceme and others do not.

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Soybean plants increase in dry weight slowly at first and then more rapidly (Borst and Thatcher, 1931). Vegetative growth ceases at about the time seed enlargement starts. The dry weights of leaves, and to a smaller extent of stems and roots, decrease thereafter, so that the total weight of the plant, including seed, at maturity is slightly less than the maximum attained 3 or 4 weeks earlier. Three distinct growth stages were recognized by Hammond and Kirkham (1949) in both greenhouse and field studies: ( 1 ) preflowering; ( 2 ) flowering and pod set; and ( 3 ) seed development. Within each stage a plot of the logarithm of weight against time gave a straight line. Relative growth rates (grams per gram per day) were about 0.085 in period ( l ) ,0.045 in (2), and 0.02 in ( 3 ) . The structure of the shoot apex and the histogenesis of the leaf have been described by Sun ( 1957b ) . The shoot apex is a low dome of meristematic cells, 80 to 120 p in diameter, containing four zones from which mature tissues arise. Differentiation of lateral leaflets begins when the leaf primordia are 140 to 200 p high. When the leaf primordia are about 380 p high, lamina1 initiation starts, Seven cell layers are formed in the leaflet blades. The first and seventh develop into epidermis, the second and third into palisade layers, and the fourth, fifth, and sixth into spongy parenchyma. The vascular system of the leaf also originates in the fourth layer. Emergence of macroscopic branches was observed by Oizumi and Katura (1958) at about the time the fifth leaf emerged. A. ENVIRONMENTAL FAIXORS The soybean plant is very sensitive to changes in its environment, and its growth responses are much less fixed than might be inferred, In its flowering response, the plant is a classic example of a short-day plant, but varieties differ in the numerical length of their effective short days (Garner and Allard, 1920, 1930). This characteristic has been used by plant breeders as the basis for classification of soybean lines first into eight and currently into ten maturity groups (Morse et al., 1949) ranging from Group 00 for Canadian latitudes to Group VIII for Gulf Coast areas. Light, temperature, moisture, root aeration, nutrient conditions, etc., all modify soybean growth qualitatively or quantitatively.

I. Light Light is of importance as the source of energy captured in photosynthesis and in the control of numerous growth processes. Light saturation of photosynthesis in soybean leaves occurs with an intensity of about 2200 foot-candles (Bohning and Burnside, 1956). Although this is only about 20 per cent of the intensity of full sunlight at midday, the intensity

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on many leaf surfaces may be less than this most of the time. The intensity under plants is greatly reduced, even in the preblooming stage, and by the time the foliage covers the row as little as 2 or 3 per cent of full intensity reaches lower and inner leaves. Reduction of sunlight to 50 per cent of normal resulted in fewer branches, nodes, and pods and only 40 per cent of normal yield (Kan and Oshima, 1952). Soybean plants subjected to artificial shading contain less nitrogen, nonreducing sugars, and starch than unshaded plants, according to Oizumi and Nishiiri (1956). After removal of shades, the levels of each of these components increased. Leaves of soybeans grown in the shade had about twice as high a N03-N percentage as those of unshaded plants (R. W. Howell, unpublished). After removal of shades the nitrate level decreased rapidly. Burstrom (1943) reported that nitrate reduction in leaves is a function of light intensity. Lower level of nitrate reductase in shaded corn plants has been demonstrated by Hageman and Flesher (1959). BILOXIsoybeans reduced nitrate more slowly on short days than on long days (Eckerson, 1932). This should probably be attributed to differences in total radiation rather than to a day-length mechanism. It is a common observation that crowded soybean plants have fewer branches than spaced plants (Williams, 1950). Individual plants adjacent to the end of a row or to a gap in the row are therefore more productive than interior plants. Plants that are shaded at about the start of seed development lose a substantial number of pods within a few days. Yamada and Horiuchi (1953) considered light to be the initial causal factor in intervariety competition. In variety composites, varieties which are taller and have the greater branching habit have a competitive advantage (Mumaw and Weber, 1957). Parker and Borthwick (1949) and Withrow and Withrow (1947) have studied the growth of soybeans under various sources of artacial light. Using a “Sunshine” carbon arc source supplemented with incandescent radiation, Parker and Borthwick obtained plant growth “equal or superior” to that in the greenhouse. Soybean plants grown under blue fluorescent light contain higher levels of protein than those receiving pink light (Howell et al., 1957). Blue light increases synthesis of organic nitrogenous compounds, according to Voskresenskaia and Grishnia (1958). The effect of light on nitrate reductase may depend on only certain portions of the spectrum. 2. Temperature

Although growth of soybeans is influenced by temperature, efforts to develop a “degree-day” function for growth have not been successful. However, D. M. Brown (personal communication) has developed a

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curve indicating a minimum temperature for growth of 50" and an optimum near 86" F. He relates rate of development ( Y ) to degrees Fahrenheit ( X ) by the following equation: Y = 4.39 X -0.0256 X2- 155.18. Growth prior to maximum podding was closely related to degree-days, but from podding to maturity, growth was more closely related to number of days than to degree-days. This agrees with the conclusion of Runge and Ode11 (1960) concerning the effect of temperature variations during seed development. Top growth is affected also by root temperature (Earley and Cartter, 1945). Height of plants was about the same at all root temperatures from 54" to go", but weight of tops was greatest with a root temperature of 81". Soybeans are fairly resistant to injury by temperature extremes. They are less susceptible to frost than corn, cowpeas, and field beans, neither young nor nearly mature plants being injured by light frost (Morse et al., 1949). They also withstand high temperatures very well, although the growth rate declines at temperatures above 100" F.

3. Soil Moisture and Aeration Growth of the soybean from germination to maturity is in general proportional to the available moisture supply, although a precise mathematical description of available moisture is difficult to make. Ueda (1952) found height, number of nodes, stem diameter, number of flowers, percentage of pod set, and number and weight of seeds, all to be positively associated with soil-moisture content. Brown and Place (1959) found that dry-weight increase in young plants is proportional to soil moisture up to the level of moisture equivalence. Soil-moisture relationships are of most practical interest when moisture extremes occur-either drought or excessively wet conditions. The soybean withstands short periods of drought without serious injury but is especially susceptible to drought injury during germination and seedling growth (Morse et al., 1949). It is less sensitive to injury by drought than corn, according to Morse et al. ( 1949). This is due at least partly to differences in the flowering patterns of the crops. Corn forms tassels and silks but once, and the coordination of these processes is of critical importance. Under drought conditions, silk development is delayed more than tasseling (Shaw, 1955) and irreparable damage results. The soybean, by contrast, flowers over a period of about a month when planted as a full-season crop. Failure of early flowers to set pods may be compensated for by better pod set or retention of later flowers. The soybean also possesses an adaptive morphological mechanism that may limit its use of water. Clark and Levitt (1956) found a higher

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concentration of lipids on the leaf surfaces of plants after exposure to drought conditions. Rates of water loss were inversely correlated with surface-lipid concentration. There was no difference in water loss between plants exposed to drought and controls after removal of the excess lipids by dipping the leaves in ethanol. Farkas and Rajhathy (1955) reported that under drought conditions the water content of lower leaves decreased more than that of upper leaves. Fukui and Ojima (1957) found that sugar content of leaves and stems increased under deficient moisture conditions whereas starch increased abnormally with excessive moisture. Nitrogen content decreased with either deficient or excessive moisture and was particularly sensitive to deficiency or excess during the flowering period. Both deficient and excessive moisture during the preflowering period retarded vegetative growth and reduced the number of flowers. When either deficient or excessive moisture prevailed during flowering, the shedding percentage increased. Hopkins et al. (1950) found that restriction of oxygen supply to the roots reduced top growth as much or more than root growth, although the soybean exhibited a “remarkable tendency to maintain growth processes” at oxygen levels in the root medium as low as 1.5 per cent.

4. Lodging Under some conditions lodging of soybeans is severe enough to reduce combining efficiency and thus to reduce harvestable yields. Although quantitative data pertaining to factors influencing the degree of lodging are limited, the lodging tendency is apparently increased by high plant populations and by conditions giving a vigorous, lush type of growth (personal communications from R. L. Bernard, E. E. Hartwig, A. H. Probst, C. R. Weber). When there is heavy rain or hail accompanied by high winds, lodging is likely to occur. Osler and Cartter (1954) found that lodging increased as planting was delayed from May 1 to June 12, but Weiss et al. (1950) found no appreciable effect of planting date on lodging of five varieties in a threeyear test at Urbana, Illinois, Ames, Iowa, and Lafayette, Indiana. Seasonal differences in factors such as temperature and moisture, which affect the amount of vegetative growth, might account for lack of agreement in the results of different experiments. Probst (1945) and Kalton et al. (1949) have shown that lodging may be severe with stands of about twelve plants per foot but negligible under the same climatic conditions with three per foot.

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B. METABOLISM 1. Photosynthesis Metabolic processes in the leaves are of special interest because here are the sites of photosynthesis. Aronoff and Vernon (1950) concluded that the sequence of formation of carbohydrates in soybean leaves is triose-,glucose-,sucrose. The isotopic labeling pattern, however, indicated that synthesis of sucrose may follow a different pathway from that of glucose. This is indicated also by data of Nelson (1956) showing that when C14-labeledglucose is applied to the petiole, the C14 remains in the glucose, but when labeled fructose is applied, the label is recovered in both glucose and fructose moieties of sucrose. Radioactive glycine, alanine, and serine were formed during a 15-second period of photosynthesis (Vernon and Aronoff, 1950), and the evidence from labeling suggested that alanine is involved in the main photosynthetic reaction. Nelson and Krotkov (1956) found alanine to be the first amino acid synthesized in soybean leaves from ammonium nitrate and C1402. Aspartic and glutamic acids were also formed, and from these asparagine and glutamine, respectively. Leaves are capable of protein synthesis (Racusen and Aronoff, 1953b, 1954) but when removed from the plant they rapidly lose this capability. A homogeneous tissue-free cell suspension fixed C02 at about one-fifth the rate that intact leaves do and also synthesized amino acids, but not protein (Racusen and Aronoff, 1953a).

2. Translocation Organic carbon compounds formed in photosynthesis are translocated little or not at all within a leaflet or to other leaflets (Aronoff, 1955). Rather, the principal movement of radioactive carbon in young plants is to the growing tips. Sucrose is probably the most important sugar in translocation. Photosynthetically assimilated C14 is translocated downward as sucrose at a velocity of about a half inch per minute (Vernon and Aronoff, 1952). The transport of sucrose, but not of glucose and fructose, is sensitive to cyanide, a respiration inhibitor (Nelson and Gorham, 195713). C14 in some unidentified form other than sugar is translocated at least twenty times as fast as sucrose, according to Nelson et al. (1958). Sugars applied to primary leaf surfaces move out more slowly in the light than in the dark. In the light, C1* tends to accumulate in stem tips, whereas in the dark the principal transport is to the root (Nelson, 1956; Nelson and Gorham, 1957a). Sugars, amino acids, and amides (Nelson and Gorham, 1957b, 1959a) are translocated readily if introduced through a cut

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petiole. Downward translocation of amino acids and amides was decreased by either excision or chilling of the roots, indicating that the roots of young plants exert a strong “demand,” which favors downward rather than upward translocation ( Nelson and Gorham, 1959b). The translocation emphasis is also downward in older plants (Belikov, 1955a,b, 1957). During pod filling the products of photosynthesis in a given leaf move mainly to seeds in the axil of that leaf. If all pods were removed from the axil of the leaf receiving radioactive COz, the concentrations of activity in seeds at lower nodes were ten to twenty times those at higher nodes. There was no translocation of Cl4O2 to other leaves. 3. Enzymes Some enzyme studies are of special interest because they reveal causative or diagnostic relationships between enzyme activity and nutritional abnormalities. Catalase activity is significantly reduced both in the genetically ironinefficient line P.I. 54619-5-1 (Weiss, 1943; Brown and Hendricks, 1952) and in normal lines (Wallace and Clark, 1956; Banerjee, 1957) when iron supply is limited. Thus a decrease in catalase activity might be a useful index of iron deficiency. Wallace and Clark also observed that in the presence of cobalt, catalase and peroxidase decreased earlier and at higher iron levels than in its absence. A possible enzymatic explanation of a type of genetic chlorosis occurring in soybean line T 219 was sought by Gage and Aronoff ( 1956). They found the activity of chlorophyllase, which destroys chlorophyll, to be unrelated to the occurrence of chlorosis. The chlorosis in that case therefore seems to be due to lack of chlorophyll synthesis rather than to its destruction. By contrast, Koski and Smith (1951) concluded that albinism in a mutant white seedling of corn arises from faster destruction than synthesis of chlorophyll. 4. Growth Substances

Marth et al. (1956) found that vegetative growth of the soybean is quickly stimulated by foliar applications of gibberellin. Wargel and Howell ( 1958) subsequently reported that increased stem elongation is observed within 2 days after spraying plants in the field. Seed treatment resulted in faster emergence, but this response was dependent on temperature, being greatest with soil temperature no higher than 70” F. Despite the responsiveness of the plant, increased yields have not resulted from either seed or foliar treatment and there appears to be little prospect of commercial usefulness of gibberellin on soybeans (Howell

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et al., 1960). Oil and protein percentages of the seed were not affected, nor was total green or dry weight of the variety VIRGINIA, when harvested as hay. The reduced growth resulting from treatment of soybeans with 2,4-D has been described by Slife (1954), and the effect of 2,4-D on various aspects of soybean nutrition by Wolf et al. (1950), Freiberg (1952), Freiberg and Clark (1952, 1955), Takijima and Hayashi (1952), and Garren et al. (1953). The effect of 2,4-D is intensified by increasing the nitrogen supply. Synthesis of protein (Freiberg and Clark, 1952, 1955) and of carbohydrates (Garren et al., 1953) is inhibited. Although the over-all effect of 2,4-D and many other herbicides is a growth inhibition, the localized effect on certain tissues may be a growth promotion, usually of a disorganized type. 2,4-D induces the pith cells in the hypocotyl to become meristematic and produce vascular strands (Sun, 1955a), but both cell division and elongation were inhibited in radicles from 2,CD-treated seeds ( Rojas-Garciduenas and Kommedahl, 1958). The herbicide, 3-( p-chlorophenyl )-1,l-dimethylurea ( CMU ), does not induce proliferation resembling that induced by 2,4-D, according to Christoph and Fisk (1954), but causes leaf epidermal cells to collapse, the palisade to become disorganized, and the mesophyll to become necrotic. Cell division is completely inhibited by benzene hexachloride (BHC), according to Sass ( 1951). Switzer (1957) reported that mitochondria isolated from soybeans 18 to 24 hours after spraying with 2,4-D had greater oxidative and phosphorylative activity than those from unsprayed controls. Key et al. (1960) presented electron micrographs showing that the 2,4-D treatment induces a growth of mitochondria. Differential toxicities of different forms of herbicides in some cases may be due to differences in ability of the plant to absorb them ( Hauser, 1955). Surfactants increased absorption rates of the salt and amine forms and decreased differences in toxicity. Similar results were obtained with corn. Meade (1958) observed increased amounts of reducing and nonreducing sugars in both corn and soybeans treated with isopropyl N - ( 3chlorophenyl) carbamate, which is an effective herbicide for grasses but is less effective on broad-leafed species. It would seem obvious, however, that primary herbicidal effects are not on gross constituents, such as sugars, but rather on substances involved in growth-controlling mechanisms at the subcellular level, such as nucleic acids, as suggested by Skoog ( 1954). The evidence recently presented by Key and Galitz ( 1959)

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that 2,4-D treatment of seedlings induces synthesis of an inhibitor occurring naturally in large quantities in immature seeds seems of special significance as to the mechanism of 2,4-D action. C. PLANTCOMPOSITION Welton and Morris (1930) many years ago determined carbohydrate and nitrogen contents of stems and leaves under low and high fertility conditions. Although total top growth was a function of fertility level, percentage composition was influenced very little. Togari et al. (1955) have shown that total weight of the plant and total nitrogen, phosphorus, and potassium continue to increase until nearly maturity. Actually, maximum total plant weight was attained about 3 weeks before maturity. After this a s,light decrease in total plant weight occurred and finally there was even a small decrease in seed weight, as previously reported by Cartter and Hopper (1942). The concentration of chlorophyll varies from 100 to 300 mg. per 100 g. leaf fresh weight, decreasing as the plants grow older (Chailakhyan and Bavrina, 1957). Carotene, initially 12 to 16 mg. per 100 g., and xanthophyll, initially 43 to 47 mg. per 100 g., also declined steadily with age. The concentrations of chlorophyll and yellow pigments in soybean leaves are also functions of calcium and magnesium supply. When calcium was high in proportion to magnesium, leaves contained relatively less chlorophyll and more of the yellow pigments (Shcherbakov, 1949, 1953a, b). Particular substances or classes of substances have been of interest to some investigators. Cohen-Boulakia (1957) found that choline increases throughout the life of the plant, except during germination. He found 0.134 mg. per plant at nodulation, 2.16 at flowering, and 5.5 at maturity, when free choline accounted for 1.8 mg. and lipid choline for the remainder. QuiIlet and Bourdon (1956) reported that maltose makes up about 45 per cent of the sugar extractable from petioles, stems, and roots, at the time of flowering and pod formation. This is surprising in view of the evidence that sucrose is the principal sugar in translocation. Cooke (1953) observed that gamma irradiation increased the ascorbic acid content of soybean and snapdragon plants. He suggested that ascorbic acid might protect plants from radiation damage, possibly by protecting enzyme systems. The average pH of leaf and stem juices is about 6, and of seeds about 6.6 (Kamae, 1957). The osmotic concentration of the juice is subject to variation due to climatic factors but gradually increases until flowering,

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and then decreases until podding, after which it again increases (Kaku, 1955a, b ) . V. Flowering

A. DAYLENGTH Our present extensive knowledge of the effects of day length on flowering goes back at least fifty years when Mooers (1908) noted a difference in the flowering period of soybeans planted on different dates. Ten years later the significance of day length in the flowering behavior of soybeans and other plants was discovered by Garner and Allard (1920). By the early 1940’s the basic facts about how light and dark periods may be manipulated to control flowering had been discovered. More recent work has been concerned with the search for evidence of a flowering hormone, with the pigments which absorb the light that controls the processes leading to flower initiation, and with rhythms and other aspects of the metabolism of flowering. The field has been reviewed frequently (Hamner, 1938, 1944; Murneek and Whyte, 1948; Parker and Borthwick, 1950; Leopold, 1951; Lang, 1952; Liverman, 1955; Doorenbos and Wellensiek, 1959). Soybeans will remain vegetative almost indefinitely if the days are long enough, or they will flower in less than a month if the days are short. Production of a macroscopic flower requires first the differentiation of a floral primordium and then the development of the primordium through various stages to the mature flower. BILOXIsoybeans will initiate floral primordia if they are given only 2 short days (8 hours of light) (Borthwick and Parker, 1938a) and are then returned to long days. If given more short days, however, more flowering nodes result (Hamner, 1940). Floral primordia are visible microscopically within 3 days and conspicuous within 10 days after the start of inductive treatment. They are first evident at the node which was second from the tip when treatment was started (Borthwick and Parker, 1938a). About 3 weeks elapse between induction and the opening of flowers in BILOXI variety (Borthwick and Parker, 1 9 3 8 ~ ).Progression from induction to flowering and fruiting requires continuation of a photoperiod of suitable length. Plants which received seven 8-hour photoperiods formed no pods if subsequently given 14-hour days ( Parker and Borthwick, 1939a). The leaf is the organ in which the flowering stimulus originates (Borthwick and Parker, 1 9 3 8 ~ ).Soybeans can be induced to flower from about the time the primary leaves appear ( Borthwick and Parker, 1938b). The most effective leaf in floral induction is the one that has most recently attained full size (Borthwick and Parker, 1940). A light intensity of at least 100 foot-candles is required (Borthwick

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and Parker, 1938c), and plants kept continuously in the dark or in dim light do not flower. If plants kept at 100 foot-candles or higher intensity for 8 hours are given an additional 8 hours of as low an intensity as 0.6 foot-candles, flowering is inhibited. This intensity, however, is merely indicative of a threshold level and higher intensities are more effective in preventing flowering (Yoshida, 1952). The number of nodes showing floral initiation is greatest on lightdark cycles of 24, 48, or 72 hours and least on cycles of 36 or 60 hours (Nanda and Hamner, 1959a, b; Blaney and Hamner, 1957). The rhythm of flowering does not appear to be related to diurnal fluctuations in auxin concentrations or in metabolism. Wareing (1953) reported that as the length of the dark period was increased, the limiting light period (longest period on which flowering occurred) for flowering was reduced. Maximum flowering occurred with a cycle of 12 hours of light and 12 hours of dark, but with an increase in the dark period to 27 hours the limiting light period fell to 10 hours. A brief period of light in the middle of a long dark period will prevent the soybean from flowering, just as though it were on a long dav (Hamner and Bonner, 1938). This has made it possible to study precisely the effect of wavelength and other aspects of the light control of flowering. The most efficient wavelength for controlling, i.e., preventing, flowering is about 6400 A and the least efficient about 4800 A. At the latter wavelength about sixty times as much energy is required for a given effect as at the former (Parker et al., 1945; Borthwick et al., 1950). The red-inhibition of flowering can be reversed by subsequently exposing the plants to “far-red” radiation (about 7350 A ) . The cycle of inhibition-repromotion of flowering by alternating exposure to red and far-Ped radiation can be repeated a number of times (Downs, 1956), but less repromotion is achieved in each succeeding cycle. It was shown by Garner (1933) that responses to day length are modified by temperature, and by Parker and Borthwick (1939b) and Roberts (1943) that temperature during the dark period is more important than that during light. Good floral initiation occurred with a leaf temperature of 65” during the dark, but none with temperatures below 55”. It was not possible to induce floral initiation in plants in a 16-hour day by temperature manipulations. Varieties of more northern adaptability probably have somewhat lower temperature responses. The primary effect of temperature is on the photoperiod reaction in the leaf blade (Borthwick et al., 1941; Parker and Borthwick, 1943). Blaney and Hamner (1957) suggested that cool temperatures may shift the endogenous flowering rhythm to a cycle length other than 24 hours.

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B. CHEMISTRY OF FLOWERING The chemistry of photoperiodism has remained until recently almost as obscure as it was at the time of the first studies of Garner and Allard, forty years ago. A number of investigators during the 1930's sought an explanation of flowering and, in fact, of photoperiodism through the carbon-nitrogen balance theory ( Kraus and Kraybill, 1918). Murneek (1937) noted an increased C:N ratio at the time of full bloom but recognized that this is much too late to be the cause of floral initiation. Parker and Borthwick (1939b) and Scully et al. (1945) reached similar conclusions. 1. Translocation of Flowering Stimulus The flowering stimulus is readily translocated up or down the plant (Borthwick and Parker, 1940) and across graft unions (Heinze et al., 1942; Galston, 1943, 1949). The rate of translocation is not reduced by a lack of oxygen in certain tissues through which transmission occurs, or by cooling stem segments to 36" to 40" F., although these treatments inhibit movement of food materials (Brun, 1954). However, Brun found no transmission of flowering stimulus through segments that had been steamed and thus presumably killed, and he concluded, as had Withrow and Withrow (1943) earlier, that transmission occurs only through living tissue. Since the xylem elements of steamed segments continued to conduct, Brun concluded that the flowering stimulus is transmitted through the phloem. As will be discussed subsequently, varieties of northern adaptation flower on longer days than BILOXI. If leaves of a variety such as AGATE (Group 00) are grafted onto BILOXI (Group VIII), the latter can be induced to flower with days too long for induction in BILOXI (Heinze et al., 1942; Galston, 1943). However, the induction period must occur after the graft is made (Carr, 1953). This may indicate that final synthesis of flowering hormone occurs in the apical meristem or that the stimulus is too labile to survive in the preinduced leaf as long as is required for development of a living graft union.

2. Hormones The existence of a flowering hormone has long been postulated and the name "florigen," proposed by Cajlachjan (1936), has been generally accepted. There is much indirect evidence for a hormone. The flowering stimulus originates in the leaves, it is translocated only through living tissue, and a single leaf can supply sufficient stimulus for the entire plant. These facts may be best explained by the synthesis of a substance(s) in

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the leaves and its translocation to sites of effectiveness in the growing regions. Most evidence on soybeans and other species indicates an inverse association of flower initiation with auxin level. In fact, Leopold states (1958, p. 283), “while most promotive effects of auxins on flowering are subtle and elusive . . . the inhibitory effects are obvious and strong.” Flower initiation in soybeans is inversely associated with auxin levels but is promoted by treatment with 2,3,5-triiodobenzoic acid (TIBA), an “anti-auxin” ( Galston, 1943, 1947). Galston’s results with TIBA were confirmed by Ishihara (1956), Fisher (1955, 1957), and Fisher and Loomis (1954). The latter authors also reported promotion of flowering resulting from spraying with nicotine sulfate and from holding growing tips down with lead weights, which presumably modified normal gravitational relationships and therefore altered auxin distribution. A transitory increase in auxin may occur at the start of induction (Cooke, 1954; Vlitos and Meudt, 1954). Application of indoleacetic acid (IAA) to plants already induced to flower hastened the opening of flowers by several days ( Galston, 1943). Promotion of floral initiation by the auxin naphthaleneacetic acid (NAA) applied for 1week prior to photoinduction has been reported by de Zeeuw and Leopold (1956). They suggested that “auxin may play a preparatory role sensitizing the plant to respond to induction treatment.” Possible interactions of auxin and temperature, and of auxin and the high intensity light reaction of floral initiation have been reported by Leopold and Guernsey (1953a, b ) and Hamner and Nanda ( 1956), respectively. Seeds soaked 24 hours in a 1 p.p.m. solution of NAA and then stored 2 weeks at 38” F. produced twice as many flowers, whereas seeds stored at 64” produced only about half as many as the controls (Leopold and Guernsey, 1953a). Hamner and Nanda found that light intensity influenced the amount of IAA required to inhibit flowering and suggested that the auxin reacts with some substance produced during the photoperiod. 3. The Light-sensitive Pignzent Borthwick et al. (1952) proposed a series of reactions to Explain known facts about wavelength characteristics of the flowering response, and Hendricks and Borthwick ( 1959) have recently summarized present knowledge of the photocontrol of various aspects of plant development. The photoreversible reaction controlling flowering is now recognized as a general reaction that also controls many other growth phenomena. Since light of different colors varies in effectiveness it follows that the substance responding to light must be colored and therefore is a pig-

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ment. The pigment can exist in two photointerconvertible forms with absorption maxima near 6600 A and 7300 A, respectively. One or the other of these forms is presumably active in the control of biochemical events leading to flowering and the other regulated phenomena. Murneek and Gomez (1936) observed that soybeans under short-day conditions were lighter in color at first but then became intensely green. Chlorophyll, however, was rejected as the active pigment by Parker and Borthwick (1950) for several reasons, most notably because of the high energy requirement, indicative of weak absorption, in the blue, where chlorophyll has a high absorption. Furthermore, leaves of dark-grown and albino plants contain little, if any, chlorophyll, but they expand rapidly after brief exposure to red light. Roberts (1948) found a decrease in carotene during the dark in soybean plants on a short day, but it appears that the carotenoids are eliminated from consideration because of the effectiveness of red light, which they do not absorb. The active form of the pigment is probably an enzyme. Some common enzymes ( catalase, peroxidase, invertase, amylase, and reducase ) were eliminated by Hibbard (1937), but Butler et al. (1959) have recently separated the pigment from living tissue by conventional methods of protein chemistry. They are now conducting studies leading to its concentration, purification, and identification. The name “Phytochrome” has been proposed for this pigment (H. A. Borthwick, personal communication).

C. OTHERASPECTSOF FLOWERING Flowering is not independent of other metabolic processes. Limiting photosynthesis either by restriction of C02 supply or shortening the period of high-intensity light reduced floral initiation (Parker and Borthwick, 1940). Eaton (1924) and Gilbert ( 1926) long ago showed that low temperature retarded flowering. More recently Klein and Leopold ( 1953) explained inhibition of flowering by maleic hydrazide as an effect primarily on growth rather than specifically on the mechanism of flowering. Langston and Leopold (1954) showed that flowering is inhibited if C02 is withheld during the dark period and suggested that dark fixation of COz might be a specific physiological factor in photoperiodic induction. If so, respiration would acquire significance as a source of COz, in addition to its general importance in supplying energy. Elliott and Leopold (1952) found that respiration of soybean leaves increased about 45 per cent during the course of three short-day cycles. Processes subsequent to initiation may be quite different from initiation in their environmental requirements. Although as few as 2 short days are required for floral initiation, progression from flowering to

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fruiting may require shorter photoperiods than the longest on which initiation occurs (Parker and Borthwick, 1939a). IAA applied after induction sped the opening of flowers (Galston, 1943) although a different auxin ( N A A ) gave only a slight increase in the number of floral primordia when applied after induction (de Zeeuw and Leopold, 1956). The shedding of flowers and young pods is increased by high temperatures and long photoperiods (Van Schaik and Probst, 1958). Kamae (1952) found no marked effect of planting rate, soil moisture, or thinning young buds on shedding of flowers and pods but observed less shedding under shortday conditions. Borthwick (personal communication to A. H. Probst, J L ~ 18, Y 1945) concluded that fertility level had no apparent effect on location of the first flower primordia. Shading plants results in both reduction of number of pods set (N. Fechtig and R. W. Howell, unpublished) and shedding of pods which are already inch long. The largest flowers are formed on a day length just shorter than that which prevents flowering (Parker and Borthwick, 1939a).

D. VARIETAL DIFFERENCES IN FLOWERING Information on specific photoperiod responses of varieties other than including those of commercial significance, is limited, but is sufficient to validate assumptions concerning the practical importance of day length in soybean production. Garner and Allard ( 1920), Borthwick and Parker (1939), Borthwick (personal communication to D. F. McAlister, Feb. 7, 1947), Parker and Borthwick (1951), Scully et al. ( 1945), Fisher and Loomis (1954), and Van Schaik and Probst (1958) have studied flowering behavior in varieties ranging in maturity from Group 0 to VIII. These studies have shown that “in general, the earlier the variety matures the longer the photoperiod on which floral initiation can occur.” Varieties of Group 0 to I11 may initiate floral primordia even on a 24-hour photoperiod ( continuous light ), but those of later maturity do not (Borthwick and Parker, 1939). It might be concluded that varieties initiating floral primordia on continuous light are “indeterminate” rather than “short-day” plants in their response to photoperiod. But floral initiation is delayed on long photoperiods and “blossoming and fruiting do not occur on photoperiods longer than 16 hours.” There appears to be no case in which a soybean variety is indifferent in its response to day length. This has been recognized by agronomists (Hartwig, 1958; Cartter, 1958) as a principal factor in determining the area of adaptation and time of maturity of varieties. Northern varieties have longer critical day lengths than southern varieties, but respond to shorter day lengths by more rapid flowering. BILOXI,

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VI. Pod and Seed Development

Soybeans are now grown almost exclusively for grain. Not since 1940 have soybeans grown for hay and similar uses occupied as much as half the soybean acreage in the United States. In 1957 all but 6 per cent of the acreage was harvested for grain (U. S. Department of Agriculture, 1947, 1958). Agronomically, then, the various aspects of growth previously discussed are of importance mainly because of their significance to seed yield and composition. Reports on the growth patterns of the pods and seeds have appeared periodically since as long ago as 1914 (Garner et ul.). Cell division in the embryo begins very soon after flowering and proceeds at an increasing rate for at least 16 days (Kato et al., 1954). Dry weight of seeds increases slowly beginning about the tenth day after flowering and rapidly about 1 week later. During the following period of about 3 weeks, when most of the dry matter is accumulated, seed weight increases an average of 6 to 7 mg. per day. A small loss in dry matter appears to be associated with ripening (Garner et al., 1914; Cartter and Hopper, 1942; Kato et al., 1954). The endosperm has a large number of dividing cells during the early stages of seed growth (Kamata, 1952) but later is almost completely absorbed and at maturity is just barely visible as the inner layer of the seed coat. Moisture content of the seed decreases throughout the period of growth (Kato et ul., 1954). Initially as high as 90 per cent, seed moisture quickly drops to 65 to 70 per cent. During most of the period of increase in seed dry weight, moisture percentage decreases veryslowly. As the seeds approach maximum dry weight, moisture percentage decreases from 60 to 65 per cent to 15 per cent or even less in 1 to 2 weeks. During most of the period of development, seed moisture content is probably not greatly influenced by normal variations in environmental conditions, but during the final period of rapid drying atmospheric conditions have a great influence on the rate of water loss (Howell et al., 1959). Togari et al. (1955) showed that the loss in weight of vegetative structures is insufficient to account for the gain in weight of the seed. C'402 introduced into an individual leaf during seed development accumulates in the seeds produced at the node of that leaf (Belikov, 1955a, by 1957, 1958). During flowering and earlier, the radioactive carbon moves mainly to growing points and young leaves (Aronoff, 1955), but during seed formation Belikov no longer found labeled carbon in either young leaves or stem tips. When the leaf at a certain node was

293 removed, seeds at that node did not develop. When the pods at a certain node were removed, the photosynthetic products were translocated mainly downward. The seeds in a given pod grow at alternating rapid and slow rates, according to Kato et al. ( 1954). In a three-seeded pod, the rapid growth period occurs fist in the apical seed, second in the basal, and finally in the central seed. Subsequently, according to Kato et al., there is a second similar cycle. A. ENVIRONMENTAL FACTORS In view of the rapid growth of the soybean seed during a relatively brief period, it is to be expected that productivity of the crop will be very sensitive to environmental conditions during this or other critical periods. The variation in a given variety between locations may be substantial, and variations between years may be greater than that between varieties within a year (Cartter and Hopper, 1942). Johnson et al. (1955) have pointed out that the genotype-environment interaction for yield is greater than for other characters of importance. Therefore, standards of good cultural practices and optimum performance of good varieties simply establish production potentials which are to be sought. Environmental variation remains a major factor affecting soybean production. PHYSIOLOGY OF THE SOYBEAN

1, Water Runge and Odell (1960) found that at Urbana yieIds were positively associated with rainfall from July 3 to July 25 and from August 12 to September 12. During the latter period, which in general coincides with the time of rapid increase in seed weight, an additional inch of rain above the average for an 8-day period resulted in a yield increase of more than 1.5 bushels. Soybeans use water from increasingly greater soiI depths as the season progresses. Swan (1959) observed water use from a depth of 51 inches, the lowest point sampled, and D. G . Hanway (personal communication) observed use from “the fifth foot.” However, Hanway noted that the greatest use of water was from the first foot of soil. The water content of the soil at the beginning of the season largely determines the amount of additional water that will be needed. In a wet season in Illinois, at the beginning of which the moisture-holding capacity of the soil was fully recharged, Swan (1959) found no significant yield reduction in plots that were deprived of further additions of water by plastic covers. Moisture losses were reduced by the covers, but it is of interest that in the dry area of central Nebraska when the land was irrigated to field capacity before planting, Hanway (personal

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communication) has obtained yields of 19 bushels or more without further irrigation. In 1955, when the 19-bushel yield was obtained, a single irrigation, filling the profile to capacity as the plants approached full bloom, resulted in a yield of about 32 bushels. A large number of irrigations (two or six), each restoring the soil moisture to field capacity, resulted in only small additional yield increases. Irrigation is essential for satisfactory soybean production in areas such as central Nebraska. In the more humid areas of the Middlewest and South, rainfall in most years is generally adequate although there may be dry periods within a season. In these areas irrigation has given less dependable results than in the West. Grissom et al. (1955), in the Yazoo-Mississippi delta, applied irrigation when the soil moisture level had fallen to 75 per cent of available capacity during seed development. Yields of 31 bushels resulted in 1952 and 41 bushels in 1954, compared with 25 and 37 bushels, respectively, on unirrigated plots. Irrigation during early stages was not beneficial. In a dry season at McCredie, Missouri, Whitt (1954) obtained a yield of 31 bushels following a single irrigation of 4.7 inches on August 21 whereas the unirrigated plot yielded only 17 bushels. Schwab d al. (1958), in a three-year study in Iowa, irrigated soybeans six times, applying 7 to 9 inches of water in all, and obtained yield increases averaging 4 to 9 bushels in four varieties. Irrigation may increase subnormal yields up to levels within the normal range when soil moisture is deficient. When natural moisture is not deficient, irrigation has not been beneficial. Above-normal soybean yields have not resulted from irrigation. 2. Temperature

The effects of temperature on yield have been studied less extensively than those of water, but it appears that temperature variations affect the composition more than the yield of seed. In their study, Runge and Ode11 (1960) found that yields were actually slightly depressed by any period of temperature above the average during July and August. In June and September above-normal temperatures resulted in small increases in yield. Increased pod and 0ower shedding occurs at high temperatures (Van Schaik and Probst, 1958), and could cause yield reductions. Takeshima (1952) reported that seed size was increased by a low minimum temperature and a large difference between maximum and minimum. The ripening period of “midseason” Japanese varieties is shortened by high temperature, but “early” varieties are little affected (Fukui and Yarimizu, 1952, 1956).

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3. Cultural Practices Cultural practices such as date and rate of planting, row spacing (width), and cultivation or other weed control measures are important in soybean production. Fertilization is discussed in the accompanying paper. The date of planting is of particular importance because of the influence of day length on flowering and other growth responses. In northern areas the soil temperature is usually not high enough for planting until sometime in May, by which time the day length is sufficiently long for satisfactory growth. In the South, however, the soil temperature becomes high enough for planting earlier in the season (Hartwig, 1958). When planting is too early, floral induction may occur and then be reversed or suspended as the length of the day increases, with unfavorable results on general productivity. In general it can be said that there is an optimum planting date for each variety and location (Weiss et al., 1950, 1952; Feaster, 1949; Osler and Cartter, 1954; Camper and Smith, 1958; Henson and Carr, 1946; Hartwig, 1954, 1958; Dimmock and Warren, 1953). Because of the desire to avoid resetting equipment that is also used on other crops, a row width of 40 inches is most commonly used by soybean producers, but studies in several States have shown that under some conditions narrower row widths are preferable (Beeson and Probst, 1955; Weber, 1953; Pendleton et al., 1960; Lehman and Lambert, 1960). The advantage of closer rows is attributed to earlier coverage of the middles by the foliage, and may also reflect more complete water use. Varieties differ in their row-width requirements. Those with “a more spreading type of growth yield best in somewhat wider rows” (Beeson and Probst, 1955). In the South, where vegetative growth is more abundant than in the North, little advantage is obtained from narrow rows (Hartwig, 1957). Thus varietal and environmental, as well as economic, factors must be considered in determining suitable row width for given conditions. Soybeans are usually drilled to obtain a stand of six or more plants per foot, but it has been shown (Probst, 1945; Weber, 1957) that gaps of at least 8 inches in the row do not reduce yield. The plants adjacent to a gap develop more branches and thus compensate for the missing plants. A method of estimating yield prior to maturity would be of value to experimentalists, to farmers, and to traders. Such a measurement might also serve as an index of nutrient or other needs of the plant. The relationship of yield to width and area of the third leaf (Kashima and Noguchi, 1953), number of seeds per pod and leaflet shape (Domingo,

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1945), and “fatness,” i.e. height times width of row coverage (J. L. Cartter, unpublished) have been studied. However, reliable “crop logging” procedures have not resulted. The close association of yield with environmental conditions during seed development makes it unlikely that any precise method of estimating yield very far in advance of maturity will be found. 4. Crop Hazards

In addition to being affected by normal variations in weather, soybean yields are also affected by crop hazards such as weeds, disease, the practices followed to control these, and such weather extremes as frost and hail. Weeds have been recognized as hazards to crop production for a long time, but only in recent years have quantitative estimates of losses due to weeds been attempted. Staniforth and Weber (1956), Weber and Staniforth (1957), and Staniforth (1958) in Iowa have studied the competitive relationships of yellow foxtail, Setaria lutescens ( Weigel) F. T. Hubb; smartweed, Polygmum pensylvanicum L.; and velvet leaf, Abutilon theophrmti Medic. with soybeans. They found yield reductions of 5 to 10 per cent due to weeds. Yellow foxtail caused slightly more yield loss than the other two weeds. In these studies weeds were planted in bands 6 to 8 inches wide over the row and the space between the rows was kept weed free. E. L. Knake and F. W. Slife (personal communication) in Illinois found yield reduced one-third in both 1957 and 1958 from a natural infestation of giant foxtail, Setaria faberii H e m . , in a 3-inch band in the row. Weeds reduced yields less in Iowa when soil moisture was limited, indicating that under moisture-stress conditions soybeans are in a favorable competitive position with weeds (Staniforth and Weber, 1956). Serious yield reductions occurred, however, when early-season moisture was followed by poor moisture conditions during the latter part of the season. Except for certain pre-emergence applications, the use of chemicals for weed control in soybeans is not recommended. Yield reductions of 40 to 90 per cent were caused by 0.1 pound per acre of 2,4,5-T applied as a spray at full bloom to 183 soybean strains (Fribourg and Johnson, 1955). C. R. Weber (personal communication) applied 2,4-D in the field to the nineteen most tolerant strains tested by Fribourg and John. son. He concluded that none of the nineteen strains had enough tolerance to justify a hybridization program to incorporate this character into soybean varieties.

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Yield and other economic characters of the seed were not adversely affected by spraying with DDT or other insecticides (Probst and Everly, 1957 ). It is generally accepted that diseases cause reduction in soybean yields, but there have been very few attempts to estimate quantitatively the extent of disease losses. It is hard to obtain artificial infections that would permit comparison of infected and uninfected plots, and when natural infections occur many factors other than the disease itself are usually involved. Recently lines that are nearly isogenic except for susceptibility to certain diseases have become available, and these appear to offer a possibility for estimating the extent of disease losses. Usin3 closely related lines for comparison, Hartwig and Johnson (1953) estimated yield losses in test plots of 8 to 11 per cent due to bacterial pustule, and Hartwig (1959) estimated yield reductions of as much as 30 per cent due to target spot. Doubtless other diseases might cause losses as large as or larger than these in small areas. However, to date there has not been reported any epidemic type of soybean disease that destroys large areas. Consequently most estimates of losses due to disease are 10 per cent or less, although loss to a particular farmer or in a small area may be as much as a complete crop failure. Nearly ripe seeds have a remarkable resistance to freezing injury. Viability of ripening seeds containing about 65 per cent moisture was reduced only slightly by exposure to a temperature of 20" F. for 10 hours (Robbins and Porter, 1946). As the seeds became drier their resistance to low temperature increased phenomenonally. After the moisture content fell to 40 per cent, exposure to -20" for 10 hours reduced viability only 50 per cent, and after moisture fell to 30 to 32 per cent, this treatment did not reduce viability at all. This is very surprising since ripening seeds with 30 to 32 per cent moisture are still respiring at an appreciable rate (Howell et al., 1959). Porter (1945) had previousIy reported reduced germination of hybrid corn seed that had been exposed to a temperature of 18 to 22" at 30 to 50 per cent moisture. Germination was not impaired by temperature in this range when moisture was 25 per cent or less. Soybean yields may be reduced by hail storms or other weather incidents, the extent of the damage depending on the severity, time, and frequency of injury. Gibson et al. (1943) repeatedly defoliated soybean plants to 0, 3, or 6 leaves at lo-, 20-, or 30-day intervals and found yields to be inversely related to severity of defoliation. Simulated hail injury (leaf removal and stem breakage) was less serious at early growth stages than later (Kalton et al., 1949; Camery and Weber, 1953; Weber, 1955). Even 100 per cent defoliation prior to blooming resulted in only a 20

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per cent yield reduction, and 100 per cent topping at the same time resulted in only 10 per cent reduction. The greatest yield reduction, more than 80 per cent, resulted from defoliation at about the time the seed started to fill. Sat0 and Nishikawa (1955) and McAlister and Krober ( 1958) reported similar results. These results illustrate the recovery ability of the plant. Defoliation by either chemical or mechanical means during seed development reduces yield, the magnitude of the effect depending on the length of time from defoliation to maturity (E. E. Hartwig, J. L. Cartter, personal communications) . Sat0 and Nishikawa (1953) found that the greatest damage from insects occurred during the early stages of seed development.

B. COMPOSITION OF SEED Soybean oil and protein have accounted for most of the demand which caused the rapid increase in soybean production beginning some twenty-five years ago, In recent years the demand for soybean meal for use in various feeds has continued strong, despite rapid increases in production, because of the excellent nutritional quality of soybean protein, Current varieties and experimental lines in the regional testing program have recently averaged about 21.5 per cent oil with a little higher in the extreme South and somewhat lower in Canadian locations. Protein has averaged 40 to 42 per cent.

1. Oil The accumulation of various chemical materials in the seed in general parallels the accumulation of total dry matter, as already discussed. However oil synthesis appears to trail somewhat behind the total gain in weight initially (Garner et al., 1914; Togari et al., 1955). The oil content increases very rapidly from the time seed weight is about 30 mg. until about 2 weeks later, after which oil percentage changes very little. The oil percentage is greatly affected by temperature during seed development. The temperature during a period of about 3 weeks beginning shortly after the start of seed development is particularly critical (Howell and Cartter, 1953, 1958). It has been demonstrated in the greenhouse that plants receiving a temperature of 85" F. during the day for as short a period as 1 week may produce seeds with oil percentages 2 or 3 points higher than plants kept at 70". This effect appears to be independent of any effect on yield. Oil content also varies with position of the seed on the plant and even with the position of the pod on a raceme. Collins and Cartter (1956) reported that seeds from the lower half of plants were 0.5 per cent higher in oil and 1 per cent lower in protein than those from the upper

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half. Seeds produced near the tip of long terminal racemes had less oil than those from points lower on the raceme. The saturated acids of soybean oil (mostly stearic and palmitic in about equal proportions) tend to be nearly constant at about 15 per cent of the total. The other acids vary, but average values are: linolenic acid 5 to 8 per cent, linoleic acid 48 to 52 per cent, and oleic acid about 26 per cent ( SchoEeld and Bull, 1944; Collins and Howell, 1957; Collins and Sedgwick, 1959; Craig and Murty, 1959). Both linolenic and linoleic acid contents decrease with increasing temperature (Howell and Collins, 1957) during seed development. The biochemistry of oil synthesis has been studied very little in either soybeans or other important oil seed crops. However such evidence as exists generally points to synthesis of the fatty materials in the cotyledons from nonfats, which are translocated in from elsewhere in the plant (Wolfe et al., 1942; Stumpf and Bradbeer, 1959). Fatty acids are probably synthesized by combining %carbon units, such as acetate, which are produced in the course of carbohydrate metabolism. Crombie and Ballance (1959) have reported evidence that acetate is the starting material for fatty acid synthesis in the fungus Trichoderma viride. Acetate also was converted to higher fatty acids by cotyledons of germinating castor beans (Coppens, 1956) and peanuts (Newcomb and Stumpf, 1953). However, net synthesis of fats is not a normal occurrence in resting or germinating seeds, and it remains to be shown that fat synthesis in developing seeds follows a similar pattern. Whether all the 18-carbon acids are produced by a common pathway, following which dehydrogenation occurs to form the unsaturated acids, or whether separate pathways exist, is not known. Simmons and Quackenbush (1954a, b ) found that seeds harvested at successive stages of maturity showed continuous increases in the amounts of saturated, oleic, linoleic, and linolenic acids. They were unable to find evidence for dehydrogenation of saturated acids, although C1* applied to sucrose appeared in oleic and saturated acids before appearing in linolenic and linoleic. Simmons (1950) earlier had suggested that linolenic acid might be necessary for synthesis of the other fatty acids since it reached its maximum level earlier. The location of fat synthesis within the cell is also unknown. Mitochondria may be involved ( Stumpf and Bradbeer, 1959). Scott ( 1955) surveyed a large number of plant tissues and reported that the fat occurred as droplets up to 1 p in diameter suspended in the protoplasm surrounding the chloroplasts and other plastids. She suggested that fat synthesis occurs within plastids and is thereafter extruded into the general cytoplasm. The fat in soybean cotyledons occurs in submicro-

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scopic droplets surrounded by a membrane which contains protein, lipoprotein, phospholipids, and nucleic acids (Kahn, 1959). It has been postulated (Kahn et al., 1958) that the membrane is of significance in synthesis of the oil as well as in its degradation. The structure of the oil molecule has been the subject of considerable study. SchoEeld and Hicks (1957) concluded that the fatty acids in soybean oil glycerides approach a random type of distribution, but Mattson and Lutton (1958) found 53% of the oleic acid on the number 2 glycerol carbon, indicating that some specificity is associated with fatty acid distribution. Vander Wal (1958) has suggested that fatty acids may “become associated by chance in those triglyceride molecules formed but are not distributed within each molecule at random.” Nontriglycerides in soybean oil amount to 5 to 10 per cent and include phosphatides, free fatty acids, sterols, and pigments (Daubert, 1950).

2. Protein According to Hammond et al. (1951) and Togari et al. (1955), the total nitrogen in the soybean plant nearly triples during seed development. About two-thirds of that in the stem, and about three-fourths of that in the leaves, in midseason is relocated before maturity. However the seed accumulation of nitrogen far exceeds that lost from other aboveground parts. Protein percentage may vary with both variety and environmental differences. It is usually inversely related to oil percentage, although not always. Protein content is less affected by temperature than is oil (Howell and Cartter, 1958) and accumulates in the seed at a more uniform rate than does oil. Protein percentage in the seed increases only slightly during development although nonprotein nitrogen percentage, which is originally high, decreases throughout development ( 0. A. Krober and R. W. Howell, unpublished). The commonly accepted factor for converting nitrogen of soybean meal to protein is 6.25. Calculations based on the amino acid distribution reported by Block and Weiss (1956) indicate the correct factor to be about 6.18. The soybean is one of the principal sources of vegetable protein for animal feeds. According to Krober (1956), the composition of soybean protein matches very closely the requirements for growth as measured with rats. Some eighteen amino acids containing about 95 per cent of the protein nitrogen, are reported in soybean protein by Block and Weiss (1956). Traces of many other amino acids are probably present. Glutamic acid is the most abundant, accounting for nearly 19 per cent of the protein nitrogen. Aspartic acid and leucine each account for about 8 per

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cent, and arginine for about 7 per cent. Methionine is the amino acid most likely to become deficient when soybean meal is used in feeds. When soybean and corn meals are used together in mixed feeds, lysine is likely to be deficient (Krober, 1956). The general field of nutritive values was reviewed by Mitchell ( 1950). Recent reports in this area have been made by Fritz (1954), Van Duyne et al. (1957), Rutherford and Pretty ( 1960), and Cohen-Boulakia (1957). There is relatively little information on the effects of environment on protein composition. Krober ( 1956) found significant seasonal variation in methionine content of Group IV varieties, but not in Group I1 or TI1 varieties. Differences due to location were inconsistent. Varietal differences were also observed by Krober and by Alderks (1949). Sheldon et al. (1951) found twice as much methionine in vegetative material from soybeans grown in nutrient solutions containing 96 p.p.m. sulfur as in those from solutions containing 16 p.p.m. Much attention has been devoted by protein chemists to the composition and physical state of soybean protein, which consists of many components (Briggs and Mann, 1950; Smith et al., 1955; Wolf and Briggs, 1956). These studies generally have not distinguished storage or nonmetabolic protein from that which is biologically the most significant, the cytoplasmic protein, including enzymes. These have been studied very little, so that the systems responsible for oil synthesis, protein synthesis, and other processes remain largely unknown in the soybean. 3. Miscelluneous

The respiratory pattern of soybean seeds during development reflects the synthetic processes in progress. Carbon dioxide production is at R maximum when oil synthesis is most rapid (Howell, 1958). Oxygen uptake under natural conditions proceeds at a uniformly low rate throughout development. However, recent experiments ( T. Ohmura and R. W. Howell, in preparation) demonstrate that the oxidative capacity of the immature seeds i s quite high and suggest that oxygen uptake is limited by physical factors such as permeability of the seed coat and pod to oxygen. Respiration continues at an appreciable rate until the seeds have dried to a moisture content of 25 per cent or less (Howell et al., 1959 ) . DiCarlo et al. (1955) found the nucleic acid content of soybeans to be about 1 per cent. This is considerably less than was found in yeast on a dry basis, but the distribution of bases was similar to that in yeast nucleic acids. The principal differences were that there was more guanine and less uracil in the soybean nucleic acid than in that from yeast.

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VII. Discussion

In reviewing the literature on soybean physiology, one is impressed by the difficulty of obtaining from existing data answers to many problems of current agronomic importance. Particularly, it is difficult, if not impossible, to determine where in the life cycle of the soybean is the process limiting productivity. This is of course not unique t o the soybean, but must ultimately appear in the study of any plant species. The soybean, if planted at appropriate spacings, intercepts about as much light, has similar water relations, and has at least as great an ability to obtain nutrients from the soil as more productive crops. Where, then, is the limitation? Is the plant inherently less efficient in transforming the elements of its environment into constituents of the seed? Does it have access, because of its growth habit, to less carbon dioxide? Is its photosynthetic system less efficient than those of other species? Is there an unsatisfied requirement for a nutrient element or water at some critical stage in the growth cycle? Do processes such as nitrogen fixation and the synthesis of oil and protein require large amounts of energy to “turn the wheels”? Is there unnecessary abortion or pod shedding? Are there genetic differences in any physiological attributes that would be useful in crop improvement? These questions are of fundamental importance to the problem of soybean productivity. Others will occur to the reader. Partial answers exist to some of them; complete answers probably to none. Investigations in all these areas if properly conceived and executed will contribute knowledge useful in soybean production. REFERENCES Akazawa, T., Funahashi, S., and Uritani, I. 1953. J. Agr. Chem. SOC. Japan 27, 849853. Alderks, 0. H. 1949. J. Am. Oil Chemists SOC. 26, 126-132. Allison, F. E., Ludwig, C. A., Hoover, S . R., and Minor, F. W. 1940a. Botun. Guz 101, 513-533. Allison, F. E., Ludwig, C. A., Hoover, S. R., and Minor, F. W. 1940b. Botun. Guz. 101, 534-549. Aprison, M. H., and Burris, R. H. 1952. Science. 118, 264. Aprison, M. H., Magee, W. E., and Burris, R. H. 1954. J. Biol. Chem. 208, 29-39 Arikado, H. 1954. Crop Sci. SOC. Japan PTOC.28, 28-36. Arnon, D. I. 1937. Soil S d . 44, 91-121. Aronoff, S. 1955. Plunt Physiol. SO, 184-185. Aronoff, S., and Vernon, L. P. 1950. Arch. Biochem. S,424-439. Asprey, G. F., and Bond, G . 1941. Nature 147, 675. Bach, M. K., Magee, W. E., and Burris, R. H. 1958. Phnt Physiol. 33, 118-124.

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Banerjee, S. 1957. J . Indian SOC. Soil Sci. 6, 169-172. Beeson, K. E., and Probst, A. H. 1955. Purdue Univ. Agr. Expt. Sta. Ext. Bull. 231, 1-39 (revised). Belikov, I. F. 1955a. Doklady Akad. Nauk S . S . S . R. [N. S.] lOa, 379-381. ( C h e m , Abstr. 49:14916, 1955.) Belikov, I. F. 195%. Fiziol. Rastenii Akad. Nauk S . S . S . R. 2:354-357. ( C h e m Abstr. 49:16080, 1955.) Belikov, I. F. 1957. Doklady Akad. Nauk S . S . S . R. [N. S.] 117, 272-273. (Biol. Abstr. 93, 15397, 1959.) Belikov, I. F. 1958. Doklady Akad. Nauk S . S . S . R. [N. S.] 120, 151-153. Bergersen, F.J. 1958. J. Gen. Microbwl. 19, 312-323. Bergersen, F.J., and Briggs, M. J. 1958. J. Gen. Microbiol. 19, 482-490. Bergersen, F.J., and Wilson, P. W. 1959. Bacteriol. PTOC., p. 25. Blaney, L. T., and Harmer, K. C. 1957. Botun. Gaz. 119, 10-24. Block, R. J., and Weiss, K. W. 1956. “Amino Acid Handbook.” C. C Thomas, Springfield, Illinois. Bohning, R. H., and Burnside, C. A. 1956. Am. J . Botany 43, 557-561. Bond, G. 1936. Ann. Botany ( L o n d o n ) 50, 559-578. Bonnier, C., and Sironvd, C. 1956. Nature 177, 93-94. Borst, H.L., and Thatcher, L. E. 1931. Ohio Agr. E z p . Sta. Bull. No. 494, 1-96. Borthwick, H. A., and Parker, M. W. 1938a. Botan. Gaz. 99, 825-839. Borthwick, H. A,, and Parker, M. W. 193813. Botan. Gaz. 100, 245-249. Borthwick, H.A., and Parker, M. W. 1938c. Botan. Gaz. 100, 374-387. Borthwick, H.A., and Parker, M. W. 1939. Botan. Gaz. 101, 341-365. Borthwick, H.A., and Parker, M. W. 1940. Botan. Gaz. 101, 806-817. Borthwick, H.A,, Parker, M. W., and Heinze, P. H. 1941. Botan. Gaz. 102, 792-800. Borthwick, H. A+, Parker, M. W., and Hendricks, S. B. 1950. Am. Naturalist 84, 117-134. Borthwick, H. A., Hendricks, S. B., and Parker, M. W. 1952. PTOC.Nutl. Acad. Sci. U.S. 38, 929-934. Briggs, D. R., and Mann, R. L. 1950. Cereal Chem. 27, 243-247. Brown, D. A., and Place, G. A. 1959. Paper presented at meeting of National Soybean Crop Improvement Council Advisory Board, St. Louis, Missouri, Aug. 9, 1959. Brown, J. C., and Hendricks, S. B. 1952. Plant Physiol. 27, 651-660. Brun, W. A. 1954. Ph.D. Thesis, University of Illinois, Urbana. Burkholder, P. R., and McVeigh, I. 1945. Plant Physiol. 20, 301-306. Burris, R. H., Magee, W. E., and Bach, M. K. 1955. Ann. Acad. Sci. Fennlcae [Ser. A, I l l . NO. 60, 190-199. Burstrom, H. 1943. Ann. Agr. Coll. Swed. 11, 1-50. Butler, W.L., Norris, K. H., Siegelman, H. W., and Hendricks, S. B. 1959. Proc. Natl. Acad. Sci. U. S. 46, 1703-1708. Cajlachjan, M. C. 1936. Compt. rend. acad. sci. U.A. S. S. 3, 443-447. Camery, M. P., and Weber, C. R. 1953. Iowa Agr. Expt. Sta. Research Bull. No. 400, 465-504. Camper, H. M., and Smith, T. J. 1958. Virginia Agr. Expt. Sta. Research R e p . NO. 21, 1-27. Carpenter, W., and Beevers, H. 1958. Plant Physiol. 33 (Suppl.), xxxv. Carpenter, W., and Beevers, H. 1959. Plant Physiol. 34, 403-409. Carr, D. J. 1953. Physiol. Plantarum 6, 672-679.

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