THE PLANT IS composed of carbohydrates, fats, nitrogenous compounds including proteins and vitamins, inorganic elements, and other substances possibly not classified in the above categories. The carbohydrates are produced by photosynthesis with the aid of a few inorganic elements such as magnesium in the chlorophyl, potassium for conversion of sugar to starch, and some of the minor elements, like manganese, for the transformation of sugar and starch to fats. The production of amino acids and protein is, however, not a photosynthetic but a biosynthetic process. This process probably depends on both the presence of, and the balance of, the supplies of inorganic elements in the plant as well as on the supply of carbohydrate. The quantities of these elements present are probably not the most important criterion of their service. Rather, the activities of the cationic and anionic constituents should probably be taken into account. It appears as if the role of these elements is mainly a catalytic one with only some of the elements actually becoming components of the protein molecule.
The primary objective of crop breeding has been that of increasing yields as measured by the quantity of material produced. Very little thought has gone to the quality of the crop in relation to the purposes it serves. This objective has resulted, in many cases, in the adoption of crops that give increased yields in carbohydrates. Yet, the provision of the carbohydrate content of rations for farm animals is a relatively simple matter. The serious problem in feeding farm animals is one of getting sufficient protein of proper quality even when we resort to the purchase of protein concentrates to balance the carbohydrates as energy in the diet.
Proteins are not commonly measured directly but only indirectly. This is done by oxidizing the organic substances slowly in the presence of sulfuric acid to retain the nitrogen. The total nitrogen in the ignited remains is determined and then multiplied by a numerical factor with the result considered as the protein. The inadequacy of such a generalized method of measuring the protein is readily evident. When it is now known that certain of the constituents of protein, namely, the essential amino acids, must be present in the animal’s diet, and when there must be a definite ratio between those essential ones in the diet for maximum efficiency in the utilization of foods, such general measures of protein by ignition and simple factor multiplication are of no service in specificity.
Chemical studies which make use of the complete destruction of the organic parts of the plant for an analysis of the remaining inorganic elements have been of some value in the qualitative analysis of the plant. But they have not enabled us to determine the combination of these inorganics with the organics produced by biosynthesis. We obtain the concentration of inorganic elements instead of their activity and function within the plant in terms of organic output.
Biological assays have been used more recently in the evaluation of feeds in relation to the fertility of the soils growing them. Little correlation has been established between the results as animal growth and the ash analyses or the protein contents as measured by the total nitrogen method. However, sufficient work has been done in feeding experiments to show that those soils generally considered poor from a viewpoint of yield production and those generally considered good show the same respective order in that crops from poor soils result in poor and diseased animals, while crops from good soils generally result in good healthy animals. It has frequently been shown that differences between fertile and nonfertile soils may be greatly magnified when measured in terms of animal growth.
This bio-assay method of evaluation, while useful along with the ash analyses of the crops, tells us nothing about different combinations of the inorganics with the organics, nor about the nature of the plant composition when any particular element in the soil is there in a deficient concentration. Therefore, we may well look to the synthetic nitrogenous compounds as good indicators of the plant processes involving cation metabolism and thereby indicating the services by the fertility of the soil. In the following study the vegetative plant parts were microbiologically assayed for their contents of nine amino acids in order to correlate, if possible, the concentration of these with the soil treatments, particularly some trace elements.
Crops, Soil Types, and Treatments
Korean lespedeza and alfalfa were the two crops assayed for their amino acids by a microbiological method as outlined by Stokes, Gunness, Dwyer, and Caswell with appropriate modifications.
The lespedeza, previously assayed chemically and biologically in feeding trials, was grown on the outlying experiment fields representing the five different soil types of Missouri. They were the Eldon sandy loam, Lintonia fine sandy loam, Putnam silt loam, Grundy silt loam, and Clarksville gravelly loam. These five soil types represent five distinctly different soil areas as regards parent material, age, topography, and vegetation. The soil treatments included lime and phosphorus on all these soils. In addition, potassium was also applied on the Eldon and Putnam soils.
The alfalfa was grown on the Putnam soil at the Missouri University farms at Columbia. Again all the plots were limed to insure a stand of the legume. In addition, phosphorus and potassium were applied as major elements. Across these plots there were applied the trace elements boron and manganese. This procedure aimed to correlate the differences found in the amino acid contents of the alfalfa with the application of the major elements and the minor elements as supplements.
Chemical Methods
The carbon determination was made by the common combustion train method and other elements were determined according to methods outlined by the Association of Official Agricultural Chemists.
Microbiological Methods
The amino acids assayed were valine, leucine, arginine, histidine, threonine, tryptophane, lysine, isoleucine, and methionine. The microbiological method begins with the hydrolysis of the plant proteins by the use of 20 ml of 5 N sodium hydroxide per gram of dried plant material for the determination of the tryptophane and 10 ml of 10% hydrochloric acid per gram of plant material for all other amino acids. A basal medium composed of amino acids, vitamins, and salts was used, and as each successive amino acid was assayed the corresponding amino acid was in turn deleted from the medium. The pure amino acid was then added to a set of standard tubes in increasing known concentrations. The protein sample was diluted by trial and error until the concentration of the amino acid in the protein sample approximated that in the standard tubes. Water was then added to the tubes to bring the volume to 5 ml and 5 ml of the appropriate basal media were added to each tube to bring the total volume to 10 ml. Each tube was then sterilized and inoculated with the proper lactic acid bacteria specific for the amino acid being assayed. After inoculation the tubes were incubated for 48 hours and then read by means of a photometer to measure the turbidity resulting from the growth or multiplication of the microorganisms in the clear media. The value (2—the log of the galvanometer reading) was plotted on the ordinate of a graph with the concentration on the abscissa. Curves were drawn for the standard and for the unknown so that the concentration of the amino acid in the unknown plant sample could be determined by reading from the unknown curve to the standard curve and then to the abscissa.
Of the lespedeza, the entire portion (that normally taken for hay) was used, while of the alfalfa, only the growing tip was taken. The reason for using the growing tip of the alfalfa was the belief that elemental deficiencies would manifest themselves first in the highly proteinaceous tip of the plant where growth represents the maximum production of protein.
Results of the Chemical Analyses & Microbiological Assays
Lespedeza Hays from Different Soil Types
The chemical analyses of the lespedeza hays suggested an order of decreasing fertility of these five soils which differed from the order established by their reputations and behaviors in crop production. The concentrations of the essential elements were higher in some cases in the hays produced on the supposedly poor soils, than in the hays produced on the supposedly more fertile soils. The percentages of nitrogen, phosphorus, and lignin increased with the soil treatments. The carbon contents of these plants were relatively constant but the carbon-nitrogen ratios varied considerably due to the variation of the nitrogen concentration from a high of 2.6% to a low of 1.9%.
The concentrations of amino acids in the lespedeza hays were extremely variable as shown in Table 1. Little, if any, direct correlation could be found with differences in soil types in that the supposedly better soils did not give the higher concentrations nor the poorer soils the lower concentrations. The concentration of the amino acids increased, in general, as the percentage of nitrogen increased but there is no direct relationship. This would indicate that possibly varying amounts of the nitrogen are combined in some of the stable ring structures or in some of the amino acids which have not been determined. This again points out the limited value of the determination of the total nitrogen and suggests the possibility of drawing erroneous conclusions when basing nutritive status upon the values for the total nitrogen alone.
The concentrations of tryptophane and threonine were apparently increased in the forage from each soil type as a result of soil treatment. The arginine concentration was higher in the hays from the treated soils in all cases except on the Clarksville, while the concentrations of the other amino acids follow no definite pattern.
A comparison of these amino acid values of the hays with the results of the biological assays of them as given by feeding tests, shows no direct quantitative agreement. The hays giving the better gains and efficiencies when fed to rabbits do not necessarily contain the higher concentrations of any of the amino acids. Instead, however, there seems to be a greater uniformity and possibly a better balance of the separate amino acids with these smaller variations among them.
Required Levels of Amino Acids for Animal Nutrition
If we compare the percentage concentrations of these amino acids with the values suggested by W. C. Rose for the concentrations necessary in the diet of the young rat for optimum growth, and if we assume these hays to be the ration, then the assayed acids, except tryptophane and methionine, were sufficient and in excess. The former was a borderline case. The latter varied considerably and was present in only about one-seventh of the amount required. These values and this observation seem valid. They are borne out by the fact that by supplementing legume hay with 0.1% of the 1-methionine, Marais and Smuts were able to increase the nitrogen utilization by young animals by as much as 30%. This certainly points to the need for supplementing the feeds—even good, green, nutritious legumes—with a protein supplement especially high in tryptophane and methionine in order to obtain optimum results.
Amino Acid Nitrogen as Part of the Total Nitrogen
When the concentrations of the amino acids were calculated as their nitrogen represented certain percentages of the total nitrogen, it was found that the nitrogen of valine, leucine, histidine, threonine, lysine, and methionine as percentage of the total nitrogen was actually decreased by soil treatment while that nitrogen for arginine and tryptophane was increased as shown in Table 2. The concentrations of the acids when expressed as per cent of the total dry weight were increased by soil treatments in most cases, as explained previously, but a smaller portion of the total nitrogen was combined in the amino acids assayed. Such facts seem rather peculiar since these amino nitrogen compounds are essential for growth and since results from feeding tests with rabbits with these same hays gave huge increases in growth as a result of soil treatment over those not treated. Evidently there is present in these treated forages some undetermined nitrogenous “growth” substance, or substances, which allows more efficient utilization of these hays than of hays from the untreated soil.
Alfalfa Hay on Treated Soils Including Trace Elements
The carbon content of the alfalfa, like that of the lespedeza, was also relatively constant. There was a rather high nitrogen content, but it varied only from 4.65% to 4.95%. The same general variation in the amino acid content of the alfalfa was found as for the amino acids in the lespedeza. The data are shown in Table 3.
The interesting observation here was that variation in the amino acid contents of these plants, which were all grown on the same soil type, was due entirely to the relatively small amount of inorganic elements applied to the soil. Probably the most significant variations were derived from the application of manganese and boron. In these two instances the greatest increase in the concentrations of most of the amino acids resulted from the manganese treatment. The exact nature of the action of these minor elements in biosynthesis is not known. It is possible, however, that their action is a catalytic one and that the proper conditions must be present for maximum efficiency. Variations resulted from the application of phosphorus and potassium, and of a mixture of phosphorus and potassium with minor elements. There was little correlation between these soil treatments and amino acid concentrations. It should be pointed out, however, that since superphosphate and potassium fertilizers contain impurities in the form of some of the minor elements, it is possible that part of the variation in the results here, as well as in practical agriculture, may be attributed to these small quantities of trace elements applied when superphosphate and muriate of potash are used.
Since plants differ only slightly in their energy values on ignition, and since the energy or the carbohydrate contents of plants are of little concern in practical feeding of farm animals, it seems that the time is ripe for some objective by the agronomists other than that of merely increasing the vegetative bulk of our crops. In the development of new hybrids, we now recognize their increased rate of depletion of the soil fertility and in many cases their lesser nutritive value per unit than for the former smaller-yielding crops they have displaced. Even in purchasing feeds we need to give more attention to the fertility of the soil upon which the crop was grown. Varieties as such are not nutritional guarantees per se. We can not safely say that alfalfa, merely because it is alfalfa, is superior in nutritional value to lespedeza or other varieties. That the nutritional values, like those in the amino acids, depend on the fertility of the soil is brought out rather forcefully by data of these studies. The results as determined by this microbiological method indicate that variations may be had in forage feeds suffering from a deficient supply of fertility elements even when that deficiency has not developed to the extent of manifesting external deficiency symptoms in the plant.
The results obtained showed that the single plant species grown on different soil types was not constant in amino acid contents and, consequently, that variations in the quality as well as the quantity of protein occurred as a result of small applications of calcium, phosphorus, and potassium on the different Missouri soils. The fact that there was no correlation between the inorganics or ash elements in the hay and the concentration of any particular amino acids, simply points out the extremely complicated mechanism of soil and plant relationship in biosynthesis. This lack of correlation also points to the extreme difficulties which arise in attempting to separate the effects of any one element in protein synthesis since the matter of synthesis concerns a balance of all the elements. A deficiency of any one, if severe enough, will disrupt the entire physiology of the plant.
1. The percentages of the essential amino acids contained in the lespedeza hays varied as a result of differences in the soil types and of differences in the soil fertility of any type as a result of soil treatment.
2. The increased concentration of the amino acids in the crop does not necessarily mean that a larger share of the crop’s total nitrogen is in the amino acid form. While soil treatments are giving higher concentrations of the amino acids there may also be an increase in the total nitrogen in the forage resulting from soil treatment.
3. Variations in the concentration of the amino acids present in the alfalfa were shown to be due to manganese, boron, phosphorus, and potassium but the minor elements manganese and boron gave the greatest increase without appreciably altering the total nitrogen.
4. It seems readily possible to improve the quality as well as the quantity of the protein produced per acre by the relatively small application and wise choice of inorganic nutrients applied to the soil.
5. The uniformity and balance of the inorganic constituents reflect themselves more in providing a relative uniformity in the amino acids than in increasing any of the individual amino acids.
6. The extremely complex nature of the soil-plant relationship renders untenable the belief in any simple evaluation of forages and other feeds by means of ordinary chemical ash analysis, and suggests that this whole subject of soil fertility in relation to both photosynthesis and biosynthesis by the crop demands further study if nutritious foods are to be continuously produced.
