l‘ I I ,I r VI IV! ,I - v $33-33?" mmémIIIIIIIIIm 'aé‘o SEED PROTEIN comm mm AMINO ACID ’ " '1 INFILTRATION OF GRAIN : RELATION TO . ‘ ND EFFECT ON SUBSEQUENT SEEDLIII'IG‘ ' ‘ : GROWTH AIIIDYIELI) * , . ‘ Thesis for the Degree‘af Ph. D. ‘ ‘ ; _ MICHIGAN STATE UNIVERSITY ‘ ; . IV w-I‘IIIIIIID IOSEPII Se“ ~ A . ' I ' ~ 197-0. ii [Ht-:5“ This is to certify that the thesis entitled SEED PROTEIN CONTENT AND AMINO ACID INFILTRATION OF GRAIN: RELATION TO AND EFFECT ON SUBSEQUENT SEEDLING GROWTH AND YIELD presented by CONRAD JOSEPH SCHWEIZER has been accepted towards fulfillment of the requirements for Ph.D. degree in Horticulture (_ . . v) ((2% ( Majtpprofessor Date 7Z/ZLZV/ //j /7 &C? 0-169 ABSTRACT SEED PROTEIN CONTENT AND AMINO ACID INFILTRATION OF GRAIN: RELATION TO AND EFFECT ON SUBSEQUENT SEEDLING GROWTH AND YIELD By Conrad Joseph Schweizer High protein seed of oats and several wheat varie- ties produced significantly larger seedlings and higher yields than low protein seed of the same genotype. There was also a positive correlation between protein content and yield which existed independent of the environmental conditions used to increase the protein content of the parent seed. Weight, per cent moisture and nitrate con- tent of the seed were not correlated with growth or yield in the following generation. In several experiments, wheat seed vacuum infiltrated for 3 hours in a 1.0 M arginine-HCl solution produced larger seedlings. In a field experiment, plants from oat seed infiltrated with arginine or a mixture of 11 amino acids yielded more grain than controls in l of 2 field tests. Etiolated wheat seedlings, from seed infiltrated with arginine, contained higher levels of total amino acids than untreated controls when harvested 6, 9 and 10 days after planting. Aspartate, glutamate and their amides were higher in seedlings developing from infiltrated seed ‘Conrad Joseph Schweizer than in controls, as expected, since they serve as central intermediates in nitrogen metabolism and transport in the germinating seed. The enhancement of seedling vigor and yield derived from high protein seed may be important in future crop production. The practicality of infiltrating amino acids into seeds to affect subsequent crop production is dif- ficult to evaluate. More importantly, these studies sug- gest that a genetic alteration in the amino acid composi— tion of storage proteins in seed may be reflected in enhanced seedling growth and yield. SEED PROTEIN CONTENT AND AMINO ACID INFILTRATION OF GRAIN: RELATION TO AND EFFECT ON SUBSEQUENT SEEDLING GROWTH AND YIELD By Conrad Joseph Schweizer A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHOLOSOPHY Department of Horticulture 1970 ACKNOWLEDGMENTS Recognition is extended to my mentor, Dr. S. K. Ries, and to Drs. D. R. Dilley, P. Filner, R. C. Herner, S. Honma, C. J. Pollard, and A. R. Putnam for their efforts relating to this thesis. I thank Dr. R. J. Laird of the International Maize and Wheat Improvement Center in Mexico and Dr. J. A. Tweedy of Southern Illinois University for supplying the wheat seed used in several experiments. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . ii LIST OF TABLES. . . . . . . . . . . . . v LIST OF FIGURES . . . . . . . . . . . . viii INTRODUCTION . . . . . . . . . . . . . 1 LITERATURE REVIEW. . . . . . . . . . . . 3 Amino Acid Metabolism During Germination and Early Seedling Development. . . . 3 Seed Protein Content: Relationship with Subsequent Growth and Yield . . . 10 Empirical Generalizations Concerning Seed Protein . . . . . . . . . . . . 12 MATERIALS AND METHODS . . . . . . . . . . 15 Seed Protein Content: Relationship with Sub- sequent Seedling Growth and Yield . . . 15 Growth Chamber, Greenhouse and Field Experiments. . . . . . . . . 15 Amino Acid Infiltration: Effect on Subse— quent Seedling Growth and Yield . . . . 17 Infiltration Techniques. . . . 17 Growth Chamber, Greenhouse and Field Experiments. . . . l9 Amino Acid Infiltration: Utilization of Arginine During Early Seedling DevelOp- ment . . . . . . . 22 General Procedures . . . . . . . . . 23 Cultural Practices . . . . . . . . 23 Analytical Procedures . . . 2A Experimental Design and Statistical Analysis. . . . . . . . . . . 25 iii RESULTS AND DISCUSSION Seed Protein Content: sequent Seedling Growth and Yield. . Growth Chamber and Field Experiments with "Genesee" Wheat. Greenhouse Experiment with "Ben Hur" Wheat. Growth Chamber and Greenhouse Experiments with "Ciano" Wheat. . . Greenhouse and Field Experiments with "Rodney" Oats . Amino Acid Infiltration. . Effect on Subse-. quent Seedling Growth and Yield Growth Chamber and Field Experiments with "Garry" Oats. . . . . Growth Chamber and Greenhouse Experiments with "Genesee" Wheat (lot II) . . Growth Chamber and Greenhouse Experiments with "Genesee" Wheat (lots III and IV) Greenhouse Experiments with "Genesee" Wheat (lot V) . . . . Greenhouse Experiments with "Inia'.' Wheat Amino Acid Infiltration: Utilization of Arginine During Early Seedling Development. CONCLUSIONS AND SUMMARY . . . . . . . . . LITERATURE CITED APPENDIX Hoagland's Solution iv Relationship with Sub- Page 26 26 26 29 31 31 38 38 A1 AA 47 “9 52 58 62 69 7O Table 10. 11. LIST OF TABLES Page Source of seed used in infiltration studies . 18 Relationship between seed protein content of "Genesee" wheat and subsequent seedling growth with and without supplemental nitrogen. 27 Relationship between seed protein content of ”Genesee" wheat and subsequent yield. . . . 27 Relationship between total amino acid content and subsequent seedling growth of "Ben Hur" wheat . . . . . . . . . . . . . . 29 Amino acid composition of protein hydrolyzates from seed of control and chemically treated "Ben Hur" wheat. . . . . . . . . . . 30 Relationship between seed protein content of Mexican grown "Ciano" wheat and subsequent seedling growth in growth chamber and green- house experiments . . . . . . . . . . 32 Relationship between seed protein content of "Rodney" oats with growth and yield the following generation . . . . . . . . . 3“ Effect of external nitrogen levels on the growth of "Garry" oat seedlings from seed infiltrated with amino acids . . . . . . 38 Effect of amino acid infiltration and nitrogen fertilization on subsequent yield of "Garry" oats at two Michigan locations. . . . . . 39 An estimate of the amount of solution infil- trated into "Garry" oat seed to account for the increase in nitrogen content . . . . . A0 Effect of seed infiltration with amino acids, purine and pyrimidine precursors on subsequent seedling growth of "Genesee" wheat . . . . 41 V Table Page 12. Effect of seed infiltration with amino acids on subsequent seedling growth of "Genesee" Wheat 0 O O I I O O O O O O O O O “2 13. Effect of amino acid and urea spray on seed- ling growth and tiller production of "Genesee" Wheat 0 O I I O O O O O O O O O O “3 1A. Effect of infiltration time with arginine and urea on subsequent seedling growth of "Genesee" wheat seed . . . . . . . . . AA 15. Effect of seed infiltration with methionine on subsequent seedling growth of "Genesee" wheat. A5 16. Effect of 1.0 M arginine infiltration and initial protein level on subsequent seedling growth of "Genesee" wheat seed. . . . . . A6 17. Effect of seed infiltration with arginine and sucrose on subsequent seedling growth of "Genesee" wheat. . . . . . . . . . . A7 18. Effect of external nitrogen levels on the growth of "Genesee" wheat seedlings from seed infiltrated with 1.0 M arginine . . . . . A8 19. Effect of seed infiltration with amino acids, sucrose and ammonium phosphate on subsequent seedling growth of "Inia" wheat . . . . . A9 20. Effect of seed infiltration with valine and methionine on subsequent seedling growth of "Inia" wheat. . . . . . . . . . . . 50 21. Nitrogen distribution in total amino acids of oats and wheat seed . . . . . . . . . 51 22. Amino acid analysis of etiolated "Genesee" wheat seedlings from control and seed in- filtrated with 1.0 M arginine . . . . . . 53 23. Amino acid analysis of ten day-old "Genesee" wheat seedlings grown in the light and in the dark from control and seed infiltrated with 1.0 M arginine . . . . . . . . . . . 5A vi Table Page 2A. Amino acid analysis of twelve day-old etiolated "Genesee" wheat seedlings and of seed from control and seed infiltrated with 1.0 M arginine . . . . . . . . . . . 56 vii LIST OF FIGURES Figure Page 1. The correlation of seed protein content with subsequent seedling growth without supple- mental nitrogen is significant at the .01 level (r = 0.81A), and with supplemental nitrogen at the .05 level (r = 0.7561) . . . 28 2. Second generation seedlings from treated "Ciano" wheat seed. . . . . . . . . . 33 3. Second generation seedlings from treated "Rodney" oat seed . . . . . . . . . . 36 viii INTRODUCTION A shift in the emphasis of agricultural technology from solely increasing crop yields to enriching the nutri— tive value of crops, as well as production, is noteworthy throughout the world. Genetic manipulation to improve the protein content and amino acid balance of agronomic crops has gained impetus since 196A with the discovery of opaque-2 mutant corn, which is high in lysine. Food pro- ducts are "tailored" with essential amino acids derived from cottonseeds, soybeans, fish protein concentrates and microbial proteins in an effort to amend the caloric in- take of some 1.8 billion people. These efforts are directed towards high biological value crops for human and animal consumption (3,7,37,A5,5A,62). It is perhaps equally important to consider the effect of alterations in seed composition on subsequent seedling growth and yield. Kidd and West (27, p. 2A9) in 1919 had a forethought: The critical question is therefore- Can we pro— pound a law to the effect that increased vigour of seedling development due to environmental conditions as distinct from hereditary causes, is correlated with increased vigour of growth throughout the life of the plant and with in- creased yield independently of the subsequent environmental conditions? They recognized that a relationship existed between in— ternal seed characteristics produced by parental environ- mental conditions and subsequent seedling vigor. Thus, they alluded to the relationship of seed protein content with subsequent seedling growth and yield. This relation- ship and the effect and utilization of amino acids infil- trated into seeds on subsequent seedling growth is the nature of the embodied thesis. LITERATURE REVIEW Amino Acid Metabolism During Germination and Early Seedling Development The idea that seed protein could serve as a nitrogen source upon germination was first advanced by Hartig (9) in 1858. It was established that these reserve materials were mobilized upon demand as simpler substances; the first, which was to be investigated, he termed "Gleis." Schulze (A9) in the late 19th century found that this substance coincided with an amino acid purified in 1806 by Vauquelin and Robiquet from asparagus Juice, called asparagine. He concluded that upon germination protein and carbohydrate degradation occurred yielding peptones, amino acids and possibly ammonia which condensed with organic acids to yield asparagine and in some species glutamine. These are mobilized and accumulate in the growing regions where they are regenerated into new pro- teins in the presence of soluble carbohydrates. Observ- ing that the nascent proteins had a different composition than those being degraded and that arginine accumulated in Pinus thunbergii seedlings, but was scant in various lupine species, he concluded that interconversion of amino acids occurred. Asparagine and glutamine acted as intermediate carriers for nitrogen which existed in the seed largely in protein. Sure and Tottingham (58) concluded from studies with etiolated pea seedlings that in the shoot region rapid carbohydrate metabolism took place during the early stages of growth. The total nitrogen of the cotyledons decreased with a concomitant increase in ammonia followed by elevated levels of a-amino acids and amides in the shoot region. Experimenters in the early l9A0's quickly re— established that nitrogen in storage protein accumulated as asparagine and glutamine in the growing regions of young seedlings. Glutamic and aspartic acids occupied central positions in protein catabolism and regeneration. The a-keto acids involved in their transamination were supplied by carbohydrate degradation (3A,61). The de— pendence of nitrogen mobilization from the endosperm upon embryo respiration was predicted by Albaum in 19A3 (1,2). Subsequent investigations focused upon the utiliza- tion of particular amino acids by excised plant parts. Oat embryos grew as well on casein hydrolyzates or on complete amino acid mixtures as on inorganic nitrogen forms (20). In deletion experiments it was found that DL-valine alone suppressed embryo growth, while in com- bination with any other amino acid it was stimulatory. L-arginine significantly enhanced root and shoot growth and increased the dry weight of the seedlings, particu- larly in combinatiOn with L-glutamate, L-lysine, DL- valine or glycine. However, in no case did they increase shoot growth when 2mM NaNO3 was added to the basal medium. Without exogenous nitrogen, germinating grain seeds showed marked increases in aspartate, alanine, glycine, lysine, and arginine (15). Concurrently, decreases were noted in glutamine, asparagine, and strikingly in proline. Folkes and Yemm (15) believed that the amino acids liberated in the endosperm were first utilized in embryo proteins, and those in excess went to other amino acids, chloro— phylls, and nucleic acid bases. Roots and sprouts of watermelon seedlings were vacuum infiltrated with labelled ornithine, arginine and urea (25). The majority of ornithine went to amino acids metabolically related, while the label in arginine was found in urea, ornithine, citrulline, lysine, histidine, proline and glutamic acid. Urea was metabolized to CO2 and NH“ which were then fixed. Apparently, the imidazole nitrogen of histidine, the guanidino group of arginine, the e-amino group of lysine, and the indolyl nitrogen of tryptophan may be utilized in the synthesis of other amino acids. Meiss (36) and others (15,31,57) also observed that maximum protein hydrolysis occurred 2A-A8 hours after water imbibition, and that mobilization of nitrogen from the endosperm was complete after 8-10 days. Wilson e£_§l. (65) in 195A found that barley seed- lings contained transaminases transferring amino groups from each of 17 different amino acids to a-ketoglutarate. Free ammonia was not an intermediate, but existed in pyridoxamine which transferred nitrogen to a-ketoglutarate in the presence of pyridoxal-S—phosphate. The main storage proteins in Vicia faba L. are legumin and vicillin, rich in basic and dicarboxylic amino acids (30). During germination there is an ex- tensive breakdown of reserve proteins with elevations in all free amino acids except arginine (6). The label in arginine—lac vacuum infiltrated into 10 day-old cotyle— dons, was found in proline and argininosuccinic acid after 5 minutes; in glutamic acid after 30 minutes; in ornithine, citrulline and asparatic acid after 3 hours; and in 11 other amino acids after 2A hours. Arginase and urease were found in roots, shoots, and cotyledons of Z, faba (23). Cotyledons of other plant species including beans and peas were found to contain labelled ornithine, proline, urea and CO2 1.5 hours after infiltration with arginine-lac. Pea seeds imbibing micromolar solutions of L- citrulline-carbamyl-luc and DL-arginine-S-luc were readily able to convert citrulline to arginine via the enzyme argininosuccinate synthetase (50). Arginine was not extensively metabolized, but incorporated as such or indirectly into proteins. Shargool and Cossins (50) concluded that arginine may serve as a storage form of nitrogen when the external nitrogen supply is high, and may be synthesized and utilized during germination when the external nitrogen supply is limiting. The nitrogen from L-arginine-U-luc injected into cotyledons of pumpkin seedlings was transported to and utilized by the growing axis (52,53). However, over 80 per cent of the carbon was metabolized to lucoa. This is contrary to evidence presented by Shargool and Cossins who found only 3 per cent luCO2 metabolized from DL- arginine-carbamyl-luc. Arginine enhanced the growth rate of pea seedlings and bud formation in tobacco stems (35). In addition, it increased the growth response of oat coleoptiles to indoleacetic acid. Sugarcane cells rapidly utilized arginine as an organic nitrogen source (35). Although the metabolism of arginine varies among species, and indeed within species, it is evident that urea cycle intermediates exist in plants and are linked to other metabolic pathways. Thus, arginine either directly or by transmination and oxidative deamination, may give rise to key amino acids localized at the site of protein regeneration. It is possible, and in some species evident (sugarcane cells and maize roots), that arginine and other amino acids (asparagine) may serve a regulatory function in the synthesis of limiting amino acids rather than be metabolized (35,AO). Asparagine, the major amide in "Genesee" wheat seed, decreased sharply upon germination with a cor- responding rise in glutamine (63). As the seedling developed, asparagine again began to predominate. Im- munochemical studies by Daussant et_al. (12) revealed electrophoretic shifts in a-arachin (storage protein of Arachis hypogaea L.) which may have been caused by pro— gressive deamidation of glutamine and asparagine. Thus, the reserve proteins not only provide amino acids but may be the first source of ammonia for the growing seedling. This study also showed that new proteins are synthesized in the cotyledons and roots of germinating seed, not identical to those of the mature seed. Those which are identical indicate resynthesis or hydration of existing proteins. Nevertheless, different proteins characterize different organs during various stages of development which exemplifies the dynamic state of amino acid inter- conversions (9,12,30,56). The fate of several amino acids and glucose added to pea cotyledons was given by Larson and Beevers (31). Glutamate and aspartate were rapidly metabolized to neutral and basic amino acids, organic acids, homoserine and 002. There was no major metabolic conversion of homoserine. Over 86 per cent of the leucine was directly incorporated into proteins of the cotyledon and later mobilized to the roots and shoots. Glucose was converted to starch, CO2 and soluble sugars. The ability of amino acids to provide carbon skele- tons for sugar synthesis was elucidated by Stewart and Beevers (57). Malate and fumarate were the principle sinks for aspartate carbon in germinating castor beans. These were readily converted to sugars with some carbon lost as CO2. Glutamine was the predominant nitrogen form transported to the growing axis. Aspartate, glutamate, alanine, glycine, and serine added to endosperm halves, were converted to sucrose before being transported. Amino acids which occupy terminal positions in nitrogen metabolism were generally transported intact to the grow- ing axis. Others occupying central positions were more‘ readily metabolized and interconverted (2A,57). The conclusions from studies on ribonuclease and protease activity during germination are not clear cut (5,15,22). Similarly, the mechanistic control of trans- aminases and amino acid interconversions in germinating seedlings is ill defined. Joy and Folkes (2A) liken con- trol to that in microorganisms. Amino acids readily metabolized provide no control, whereas those not metabo— lized are blocks in synthesis. Oaks (A0) working on asparagine transport in maize root contended that amino acid requirements at the growing axis may regulate 10 protein degradation, since externally supplied amino acids delayed the disappearance of nitrogen from the endosperm. Asparagine may have been transported accord- ing to supply and demand in the root tip. Undoubtedly, the rate of degradation of storage proteins and carbo- hydrates is in some way under control by the growing axis. Many species of the family Fabaceae colonize soils deficient in nitrogen (6). Their reserve proteins are high in basic amino acids, particularly arginine. These may supply the basic nitrogen components for other amino acids, nucleic acids, prosthetic groups etc. necessary to maintain the developing photosynthetic and respiratory apparatus of the germinating seed, until the seedling can manufacture its own. Seed Protein Content: Relationship with Subsequent Growth and Yield Experimentation directly relating seed protein content with subsequent seedling growth and yield is negligible. Galligar (16) in 1938, observed the growth of root tips in sterile nutrient cultures from seed of sunflower, cotton, pea, castor bean, corn, and soybean which varied in protein and carbohydrate content. Root tips from seed high in starch grew well, whereas those from high protein seeds were less able to maintain growth. Seeds high in sugar and oil had varied root growth among species. It is important to recognize that the negative ll relationship between the seed protein content and subse— quent root growth was observed between plant species and not within a single species. Also, enhanced root growth is not necessarily correlated with favorable shoot growth and yield. Relationships of seed size with subsequent seedling growth and the effect of seed treatment with trace ele- ments, nitrogenous compounds and other materials on yield, carbohydrate and protein content of grain are well docu- mented (8,10,11,17,18,21,26,28,38,51). In general, larger seeds produce larger plants and higher yields; those where part of the endosperm had been progressively re— moved, produced smaller seedlings. No correlation was found between the carbohydrate reserves in white mustard seeds and hypocotyl length (55). However, a positive correlation was found between hypocotyl width and carbo- hydrate content of seeds from the same population. A reciprocal relationship was found between germination ability and protein content in wheat, rye, barley and oat seeds (AA). The alteration of seed protein and carbohydrate content due to fertilization and other environmental conditions is extensively reviewed (ll,l7,A6,A7,A8,59, 62,6A). Those practices which increase crop yields generally lower the protein content of grain. Some workers express doubt that maximum yield and protein can 12 be achieved in a single species through cultural prac- tices and/or breeding (39). Soaking and coating seeds with various biologically active compounds has produced diversified results in many crops (8,18,21,38,51). Removal of seed coats resulting in the loss of amino acids, carbohydrates and other water soluble materials during imbibition has led to reduced germination and seedling growth (32). Hypothetically, seed coat removal in those species which do not exhibit seed coat dormancy results in rapid imbibition, disrupt- ing membrane integrity,allowing diffusion of soluble solutes. The nutritive value of seed proteins in animal consumption is abundantly manifest in the literature. Feeding studies often confirm that germinated seeds are higher in biological value than ungerminated seeds (3A). Dietary essential amino acids may be synthesized upon germination improving the nutritive value of the seed. Empirical Generalizations Concerning Seed Protein 1. Upon germination reserve proteins and carbo- hydrates are degraded to amino and organic acids which are mobilized to sites of reorganization at meristematic regions. l3 2. In seeds, nitrogen exists mainly in storage proteins which are utilized by the germinating seed, re- gardless of the external nitrogen supply. 3. Storage proteins of many plant species are rich in basic amino acids, particularly arginine, capable of being oxidatively deaminated or transaminated to d- keto acids. The loss of protein from the endosperm is reciprocally related to protein increases in the embryo. A. Glutamate, asparatate and their amides occupy central positions in protein degradation and resynthesis. 5. The enzymatic machinery necessary to metabolize exogenously supplied amino acids is evident in germinat— ing seeds. In particular, enzymes and urea cycle inter— mediates are present in various seed organs incubated with arginine indicating its possible metabolism to other amino acids. 6. A diverse variety of new proteins are present in the germinating seed exemplifying the dynamic state of amino acid metabolism. 7. The degradation of storage materials appears to be controlled by the meristematic axis, possibly in— volving a type of substrate feedback inhibition. 8. Within a species, larger seeds produce larger seedlings and higher yields. 1A 9. In general, environmental parameters affect- ing crop yields reciprocally effect their seed protein content. 10. In the limited studies recorded, seed protein content of various plant species is negatively correlated with subsequent root growth and per cent germination. MATERIALS AND METHODS Seed Protein Content: Relationship with Subsgquent Seedling Growth and Yield Growth Chamber, Greenhouse and Field Experiments Winter wheat (Triticum aestivum L., cv. Genesee) was obtained from a 1967-68 commercial planting in south- western Michigan. The enhanced seed protein content was due to postemergence subherbicidal applications of simazine [2-chloro-A,6-bis(ethylamino)-s-triazine] or terbacil [3-tert-butyl-5-chloro-6-methyluracil] (A7). Seed was sown in 8 x 10 cm plastic cups in vermiculite in a growth chamber maintained at 10°C during the night and 20°C during the day (16 hours). Each of the 3 field replicates was again replicated 2 times. Equal volumes of Hoagland's solution (Appendix I), with the addition or deletion of supplemental nitrogen, were added to each cup as needed. Hoagland's solution containing 3 mM NO3- was added 22 days after planting. Plants not receiving nitro- gen were harvested 27 days after planting and those re- ceiving nitrogen were grown for A0 days. Each of the 3 field replicates was again replicated 3 times in a field experiment in central Michigan on 15 16 September 13, 1968 and harvested July 22, 1969. Eighty grams of seed was planted per 1.2 x 5.5 M plot. Winter wheat ("Ben Hur") used in a greenhouse study was obtained from a field experiment at Southern Illinois University in 1968. The increased protein content of the seed was due to soil surface applications of simazine or atrazine [2-chloro-A-(ethy1amino)-6-(isopropy1amino)-s- triazine] plus a nitrogen application of 35 kg/ha. Samples from each of the 3 field replicates were again replicated 3 times in a greenhouse maintained at 20-27°C. Seed was planted in 18 cm clay pots in a sandy clay loam. Plants were grown for 50 days and watered as necessary. In growth chamber and greenhouse experiments, spring wheat ("Ciano") was obtained from a 1968 experiment con- ducted at Ciudad Obregon, Sonora, Mexico. The varying seed protein content among treatments was in response to nitrogen applications. Treatments were replicated 3 times in the growth chamber and watered as necessary with Hoagland's solution containing 3 mM NOB'. Three replicates for each treatment were made in the greenhouse study; other environmental parameters were as previously des- cribed. Plants in the growth chamber were harvested after 31 days and those in the greenhouse after Al days. Oat seed (Avena sativa L., cv. Rodney) from a 1967 field experiment in central Michigan was planted in the field on April 18, 1968 and the grain harvested July 30. 1? Forty grams of seed was planted per 0.61 x 9.2 M plot from each control, simazine and terbacil treatment lot, and each seed lot was replicated 3 times. Plots were periodically hand hoed as a weed control measure. Similar seed was also sown in clay pots in two greenhouse studies each containing 3 replicates. Environ- mental parameters were as previously defined; one experi- ment extended 19 days, the other 2 months. Amino Acid Infiltration: Effect on Subsequent Seedling Growth and Yield Infiltration Techniques The source of seed used in these experiments is described in Table 1. Seed was added to L-amino acid solutions at a ratio of A grams of seed per 10 ml solu- tion. The pH of the solutions was not determined. Flasks containing the seed and solutions were placed in a dessicator and a vacuum of 730 mm mercury was applied by a water aspirator for 3 hours with 5 periodic releases. Solutions were immediately decanted upon removal from the dessicator, and the seeds rinsed twice with distilled water, blotted dry and placed in a drier at A5°C for 2A hours. Total nitrogen was determined on samples taken from each treatment. TABLE l.--Source of seed used in infiltration studies. 18 l Lot Number and Kind Cultivar Source and Comments II III IV VI Oats Winter wheat Winter wheat Winter wheat Winter wheat Spring wheat Garry Genesee Genesee Genesee Genesee Inia MSU Seed Foundation Lab; visually sorted. Untreated controls from 3 replicates of the 1967-68 experiment in southwestern Michigan; visually sorted. Untreated controls from 3 replicates of another 1967- 68 field experiment in southwestern Michigan; visually sorted. Untreated controls from 3 replicates of a 1967-68 experiment in central Michigan; visually sorted. Untreated controls from a 1968-69 experiment in southwestern Michigan; screened through a 6 1/2 / 6A mesh screen, smaller seed discarded. Untreated controls from a 1968-69 experiment at Obregon, Mexico; screened through a 6 1/2 / 6A mesh screen, smaller seed dis- carded. 1Each lot of seed was thoroughly mixed before use. 19 Growth Chamber, Greenhouse and Field Experiments Oat seed from lot I (Table 1) was infiltrated with a solution of 0.67 M arginine-H01 and a solution contain— ing alanine, arginine-H01, asparagine, glycine, glutamine, histidine, leucine, lysine-H01, methionine, proline and glutamic acid-HCl each at 0.09 M. Samples from each treatment were germinated in vermiculite moistened with distilled water, in a growth chamber at 27°C. After 6 days plants were transferred to nutrient cultures of Hoagland's solution with 2, 8 or 16 mM NO3-. Six plants were grown per 200 ml jar lined with aluminum foil, with 3 replicates per treatment, in a growth chamber main- tained at 15°C during the night and 20°C during the day (16 hours). Solutions were changed every 2 days and the plants were harvested after 2 weeks. Seed from the same treatments was used in field experiments at 2 locations in Michigan. Forty grams of seed was planted per 0.61 x 9.2 M plot with a main nitrogen split of 0 and A5 kg/ha, with 3 replicates. In south- western Michigan, seed was planted April 11, 1969 and harvested July 25; in northern Michigan, seed was planted May 3, 1969 and harvested August 20. Plots were period- ically hand hoed as a weed control measure. Wheat seed from lot II was infiltrated in solutions of amino acids, purine and pyrimidine precursors and 2O grown in a growth chamber for 2 weeks under conditions previously defined. Four replicates per treatment were grown in vermiculite and watered as necessary with Hoag- 3 Solutions of 1.0 M glycine + 1.0 M alanine + 0.25 M land's solution containing 8 mM NO glutamic acid—HCl, 0.5 M histidine, 1.0 M arginine-RC1 and 0.25 M valine were used in infiltrations of similar wheat seed from lot II. Seed was sown in clay pots and 5 replicates were grown in a greenhouse for 1 month under environmental parameters previously defined. In another greenhouse experiment, 2 week-old wheat seedlings (from lot II) were sprayed with a mixture of amino acids or urea. The A replicates of 10 plants per pot were harvested after 20 days. Wheat seed from lot III was infiltrated with arginine-HCl and urea for l, 3 and 9 hours with the vacuum released 5 times during each time interval. Four replicates were grown in a greenhouse under conditions previously defined and harvested after A5 days. An infiltration solution of 0.3 M methionine was used with wheat seed from lot III. Seed was sown in clay pots in a sandy loam and each treatment was replicated A times in a greenhouse. Plants were watered as neces- sary and harvested after 1 month. Wheat seed from lots III and IV infiltrated in a 1.0 M arginine-H01 solution was grown in clay pots in a 21 sandy loam in a growth chamber maintained at 10°C during the night and 20°C during the day (16 hours). Plants were watered as necessary with Hoagland's solution con— taining 3 mM NO3- and the A replicates were harvested after A2 days. Wheat seed from lots V and VI was infiltrated in solutions of 1.0 M arginine-H01 and 1.0 M arginine-HCL + 0.5 M sucrose. Seed from lot VI was also infiltrated in solutions of valine, glycine + alanine + glutamic acid- HCL, ammonium phosphate and methionine. Twelve seeds were planted per pot in a clay loam and randomly thinned to 10 seedlings upon emergence. These greenhouse experi- ments, each employing A replicates, were watered as necessary and harvested after 1 month; except one experi- ment with "Inia" wheat which was grown for A2 days. Infiltrated seed from lot V was germinated in perlite moistened with distilled water, in a growth chamber at 27°C. After 1 week the seedlings were trans- planted to nutrient cultures of Hoagland's solution con- taining o, A, 8, or 16 mM No3', with 3 replicates. Solu- tions were changed every 2 days and the plants were grown for 2 weeks in a greenhouse. Controls infiltrated with distilled water and non— infiltrated controls were planted from each treatment lot in all respective experiments. 22 Amino Acid Infiltration: Utilization of Arginine During Early Seedling Development Control and 1.0 M arginine-RC1 infiltrated wheat seed from lot V was surface sterilized with 1% sodium hyperchlorite and planted in perlite, moistened with dis— tilled water. Seeds were germinated in a growth chamber maintained at 27°C, in the dark for 6, 9, 10 and 12 days. After 6 days, the top A cm of 50 harvested uniform seed- lings, including coleoptiles, were immediately ground to homogeneity in A0 ml of distilled water at A°C with a mortar and pestle and then freeze dried. Similarly, the top 12 cm of 30 uniform seedlings harvested after 9 days were ground at A°C and freeze dried. The top 7 cm of 60 uniform seedlings were har- vested after 10 days and the top 12 cm of 50 uniform seedlings were harvested after 12 days, similarly ground at A°C and freeze dried. The tissue harvested from 9, 10 and 12 day-old etiolated seedlings did not include coleoptiles. Seed was also grown in a greenhouse maintained at 20-27°C, in the light. The top 6 cm of 60 uniform seed— lings were harvested after 10 days, similarly ground at A°C and freeze dried. Twenty or A0 mg samples from the freeze dried material were extracted 3 times in gently boiling 70% ethanol for 15 minutes. The supernatant solutions were 23 combined, evaporated to dryness, and dissolved in lithium citrate buffer pH 2.8 for subsequent free amino acid determinations on an amino acid analyzer (A2). The hydrolyzate of 10 mg of tissue from each of the samples was used in total amino acid analysis. Twenty grams of ungerminated seed, ground through a A0 mesh screen by a Wiley mill, was also freeze dried and total and free amino acids determined by the outlined procedures. Homogeneity of the freeze dried material was checked by taking several 10 and 20 mg samples for total nitrogen determinations. General Procedures Cultural Practices Unless otherwise specified, in growth chamber and greenhouse experiments, 20 uniform seeds were planted in each pot and randomly thinned to 15 seedlings upon emerg— ence. In all growth chamber and greenhouse experiments plants were immediately weighed upon harvest and oven dried at A5°C for 72 hours and then reweighed. Field experiments had standard guard rows and alleys which were removed before harvest. Seed heads were threshed with a small plot threShing machine, air dried and cleaned before analysis. 2A Analytical Procedures Twenty gram samples ground with a Wiley mill through a A0 or 60 mesh screen were uniformly dried and used for total nitrogen, nitrate and amino acid determinations. Total nitrogen was analyzed by micro-Kjeldahl or auto- matic procedures (A,13,60). Samples for the nitrogen analyzer were pre-digested in sulfuric acid. Perchloric acid and selenium were added to the mixture prior to digestion and distillation. Ammonia was determined by a color reaction with alkaline phenol and sodium hypo- chlorite. Total nitrogen was estimated by optical density standardized by micro—Kjeldahl analysis. A factor of 5.7 for wheat and 6.25 for oats was used in the conver- sion of total nitrogen to protein. Nitrate was determined according to Lowe and Hamilton (33). Amino acid composition of protein hydroly- zates was determined on an amino acid analyzer (l9,A2). Samples were hydrolyzed in evacuated tubes containing 6 N HCl (glass distilled) for 22 hours at 110°C (20 psi). After hydrolysis, the samples were evaporated to dryness and dissolved in lithium citrate buffer pH 2.8 for subse- quent total amino acid determinations. To determine seed weight 3 or 5 replicates, each containing 100 or 200 uniform seeds, were oven dried at A5°C for 2A hours and weighed. Per cent seed moisture 25 was determined by drying 3 replicates, each containing 100 uniform seeds, at 102°C for A8 hours. Experimental Design and Statistical Analysis Treatments were arranged in randomized complete blocks in all single factor experiments. A split-plot design was employed in all factorial experiments with nitrogen levels as main plots and treatments as sub- plots. All experimental data was subjected to analysis of variance. Trend comparisons were made with single de- grees of freedom. Tukey's test was used to compare means when a significant F value for a factor was obtained. Correlations were made wherever relevant. RESULTS AND DISCUSSION Seed Protein Content: Relationship with Subsequent Seedling Growth and Yield Growth Chamber and Field Experiments with "Genesee" Wheat The effect of subherbicidal-treatments on increasing the seed protein content of legumes and grain crOps has been reported (A6,A7). The protein content of wheat seed as measured by micro-Kjeldahl analysis was positively cor- related with subsequent seedling growth (Table 2). Seed- ling growth was more closely associated with seed protein content when the environmental nitrogen supply was low (Figure 1). There was no significant correlation between seed nitrate and seedling growth, and the per cent dry weight of the forage was not altered. In a field experiment,the enhanced protein con— tent of the seed (based on Kjeldahl N) due to chemical or nitrogen applications was significantly correlated with subsequent yield (Table 3). The higher seed pro- tein content in the first generation was not reflected in higher seed protein content in the second generation. This is logical, since inheritance of adaptive alterations in response to environmental conditions is unsubstantiated. 26 27 TABLE 2.--Relationship between seed protein content of "Genesee" wheat and subsequent seedling growth with and without supplemental nitrogen.1 Growth Rate Seed Analysis Without N With N Chemical (kg/ ha) Total Nitrate Fresh Dry Fresh Dry Protein wt wt (muM/g wt wt (mg/8 dry wt) (mg/ (z) (mg/ (%) dry wt) plant) plant) Control 0 96 a 303 85 27 119 a 26 Simazine .56 102 ab 337 88 26 126 ab 26 Terbacil .28 108 b 265 97 27 132 b 26 lMeans followed by unlike letters are significantly different at the .05 level. TABLE 3.——Re1ationship between seed protein content of "Genesee" wheat and subsequent yield. First Generation Second Generation Seed Seed Rate Yield N level protein protein (kg/ha) Chemical é:%/ (mg/g A231 (mg/g dry wt) dry wt) 0 None 0 96 A032 10A Simazine .56 102 3731 10A Terbacil .28 108 3951 105 Mean 102 3905 a 10A8 22 None 0 106 3808 11A Simazine .56 119 AlAO 113 Terbacil .28 120 A2A3 96 Mean 115 A06A ab 108 A5 None 0 128 A301 95 Simazine .56 1A6 AA18 106 Terbacil .28 139 AA09 105 Mean 138 A376 b 102 1 Means followed by unlike letters are significantly different at the .05 level. The correlation between seed protein content and subsequent yield is significant at the .01 level (r = 0.88A). 28 .A©m>.o u av Hm>mH mo. map as :omoppfi: HmpcmEmHQQSm he“; see .Aaflm.o u av Hc>cfi Ho. the he ececaaaemAh ha cowopufic HmucmEmHQQSm usocpfis nuzosm wcfifipmcm pcmsomeSm spas peopcoo cmfipoaa econ go coapmamnsoo mzeII.H msswfim 6:5 :3 {95 2.30: on; m: o: no. no. no Z al‘pq.w.zm.a..mqam I. [III 2 a Seed Growth analysis Conc ———————- Dry wt Treatment (M) Total N (ms/s Planti/ (ms/2 (ms/ 1 (5) dry wt) pot pot) plant) Control - 19.1 1A.8 a 2A32 a 16A a 23.2 Water 0 19.8 15.0 a 2A28 a 162 a 23.3 Histidine 0.5 22.5 1A.8 a 2675 a 181 a 23.2 Arginine 1.0 29.0 1A.2 a 2892 b 205 b 23.2 Valine ‘ 0.25 20.5 15.0 a 2775 a 185 a 23.5 Glycine, 1.0 ' alanine, 1.0 glutamic acid 0.25 23.A 10.2 b 2331 a - 21.9 1 Means followed by unlike letters are significantly different at .01 level. 2Means followed by unlike letters are significantly different at .05 level. A3 A single application of an amino acid mixture or urea on the foliage of 2 week old wheat seedlings pro- duced an increase in both growth and tiller production (Table 13). Each treatment solution contained equivalent amino groups. Foliar sprays of urea have been shown to increase the yield and protein content of "Pawnee" wheat by acting as a nitrogen source (1A). Similarly, the amino acid mixture may function as a source of ammonia for the synthesis of proteins at the growing axis. TABLE l3.--Effect of amino acid and urea spray on seedling growth and tiller production of "Genesee" wheat. Growth Treatment Conc ' (M) (mg/plant) _ Tillers Fresh wt Dry wt x/10 plants Control - 836 a 150 ab 1.3 a Water 0 813 a 1A6 b 2.0 a Arginine, 0.5 methionine and 0.2 glutamic acid 0.3 1126 b 185 ac 12.0 b Urea 1.25 1180 b 201 c 1A.3 b Urea and , amino acids 1/2 above 1080 b 191 c 12.8 b' lMeans followed by unlike letters are significantly different at the .01 level. AA Urea infiltrated into "Genesee" wheat seed however, did not increase the subsequent seedling growth as well as arginine (Table 1A). TABLE 1A.-—Effect of infiltration time with arginine and urea on subsequent seedling growth of "Genesee" wheat seed.l Infiltration time (hr) l 3 9 Cone Treatment (M) N N N Fresh Fresh Fresh (me/s (mg/s (mg/g dry wttgg/ dry wtt(g/ dry wt (g/ wt) p0 wt) p0 ) wt) p0t) Control 15.3 5.62 15.3 5.21 15.3 5.09 Water 1A.9 5.50 15.8 5.60 13.7 A.97 Arginine 19.7 5.16 21.A 6.15 22.0 5.53 Arginine 2A.6 5.5A 27.0 6.15 2A.9 6.03 Urea 17.9 5.A0 19.8 5.61 19.6 5.63 Urea 21.8 5.9A 23.A 5.90 23.A 5.62 1 F value for interaction of treatment with time is significant at the .05 level (CV = 10.7%). Growth Chamber and Greenhouse Experiments with "Genesee" Wheat (lots III and IV) An infiltration period of 3 hours was determined as optimum based on a greenhouse experiment with wheat seed (Table 1A). The vacuum was released 5 times during each time interval. The nitrogen content and the subsequent A5 growth of the seeds infiltrated for 9 hours shows little benefit over 3 hours. The low nitrogen content of the water infiltrated control after 9 hours indicates leaching of solutes from the seed. After this period of time the seeds were actively germinating and may exclude further infiltration of solutes. The per cent dry weight of the forage was not altered. In another greenhouse experiment with wheat seed from lot III, the subsequent seedling growth was reduced due to methionine infiltration (Table 15). The per cent dry weight of the forage was not altered. TABLE 15.-—Effect of seed infiltration with methionine on subsequent seedling growth of "Genesee" wheat. Seed analysis Growth-dry wtl Treatment Efigc Total N (mg/g dry wt) (ms/plant) (%) Control - 15.0 82 a 16.3 Water 0 15.6 86 a 16.5 Methionine 0.3 1A.A 63 b 16.2 1Means followed by unlike letters are significantly different at .05 level. The difference in protein content of wheat seed from lots III and IV was due to locations (Table 1). Subsequent seedling growth from low protein seed (lot 111) A6 infiltrated with arginine was significantly greater than controls (Table 16). High protein seed (lot IV) similarly infiltrated with arginine did not enhance seedling growth. Arginine infiltration into wheat seed was of little ad- vantage when the inherent protein content was high. It is likely that the smaller, high protein seeds were not able to supply sufficient a-keto acids during the early stages of growth to utilize the additional ammonia from the infiltrated arginine. TABLE l6.--Effect of 1.0 M arginine infiltration and initial protein level on subsequent seedling growth of "Genesee" wheat seed. Seed parameter Low protein seed High protein seed Wt (me) 33.0A 25.50 Moisture (%) 8.7 7.1 Total pro— tein/seed (mg) 2.87 3.3A After infil— tration: Control Water Arginine Control Water Arginine Seed N (mg/g dry wt) 15.2 14.2 21.5 23.0 22.7 3A.7 Dry wt (mg/ plant)1 71 a 80 a 103 b 86 81 88 Dry wt (%) 2A.9 2A.3 25.9 25.2 25.2 2A.8 lMeans followed by unlike letters are significantly different at the .01 level. A7 These data also show that high protein seeds pro- duce larger seedlings and that this relationship is not a positive function of seed weight or per cent moisture. The per cent dry weight of the forage harvested was not different between treatments. Greenhouse Experiments with "Genesee" Wheat (lot V) Sucrose added to an infiltration solution of arginine was less effective in stimulating growth than arginine alone (Table 17). A 1.0 M arginine solution again produced a significant increase in subsequent seed- ling growth. Sucrose may have competed with arginine for entry into the seed as evidenced by lower total nitrogen per seed. The per cent dry weight of the forage was not changed. TABLE l7.—-Effect of seed infiltration with arginine and sucrose on subsequent seedling growth of "Genesee" wheat. Seed analysis Growthl Treatment Conc (M) Total N Fresh wt Dry wt (mg/g dry wt) (ms/plant) (%) Control - 16.1 685 a lA.7 Water 0 16.1 723 a 1A.7 Arginine 1.0 20.7 816 b 1A.5 Arginine and 1.0 sucrose 0.5 19.1 '7A3 ab 1A.8 lMeans followed by unlike letters are significantly different at the .01 level. A8 Arginine infiltrated into wheat seed affected seed- ling growth when an external nitrogen supply of 8 or 16 mM N03- was supplied (Table 18). The effect was optimum at 8 mM N03", which was similar to previous results with oats (Table 8). TABLEliL--Effect of external nitrogen levels on the growth of "Genesee" wheat seedlings from seed infiltrated with 1.0 M arginine. Seed 1 analysis Growth (mg dry wt/plant) Conc Treatment (M) Total N mM N03- (mg/g dry wt) 0 A 8 16 Mean Control - 16.1 27 132 127 130 10A Water 0 16.1 27 130 129 127 103 Arginine 1.0 20.7 28 132 1A7 1A0 112 Mean2 27 a 131 b 13A b 132 b 1 Means followed by unlike letters are significantly different at the .05 level. F value for interaction of linear nitrogen with control vs. arginine is significant at the .05 level (CV = 6.7%). 2Means followed by unlike letters are significantly different at the .01 level. These data indicate that there is a basic nitrogen level necessary for optimum utilization of arginine at a particular stage of growth. At extremely high or low nitrogen levels the effect of arginine on 3 week old A9 seedlings is minimal. This is logical, since arginine cannot be infiltrated into the seed in a quantity neces- sary to supply all the nitrogen for cellular metabolism throughout the life of the plant. The per cent dry weight of the forage harvested was not changed between treatments within a nitrogen level. Greenhouse Experiments with "Inia" Wheat Mexican wheat seed infiltrated with various amino acids, sucrose and ammonium phosphate did not signifi- cantly produce larger seedlings than controls, although the total nitrogen content of the seed planted increased due to treatments (Table 19). A mixture of glycine, alanine and glutamic acid did not reduce germination as it did in a previous wheat variety (Table 12). TABLE 19.--Effect of seed infiltration with amino acids, sucrose and ammonium phosphate on subsequent seedling growth of "Inia" wheat.l Seed analysis Growth—dry wt Treatment Conc (M) Total N (mg/g dry wt) (ms/plant) (%) Control - 18.1 355 20.1 Water 0 18.8 357 19.6 NHuH2POu 0.5 2A.6 351 18.9 Arginine 1.0 2A.6 363 19.1 Arginine and 1.0 sucrose 0.5 2A.2 385 19.2 Glycine, 1.0 alanine and 1.0 glutamic acid 0.25 22.8 35A 19.6 lF value for difference in treatments is not signifi- cant. 50 Seedling growth from seed infiltrated with valine or methionine did not differ from controls (Table 20). Methionine significantly reduced the growth of "Genesse" wheat in a previous experiment (Table 15). In both ex- periments with "Inia" wheat the per cent dry weight of the forage harvested was not altered. TABLE 20.--Effect of seed infiltration with valine and methionine on subsequent seedling growth of "Inia" wheat. Seed analysis Growth-dry wt Treatment 7880 Total N l (ms/a dry wt) (mg/plant) (%) Control - 18.1 216 15.6 Water 0 19.3 20A 15.2 Valine 0.25 18.1 210 15.8 Valine 0.50 20.A 206 15.2 Methionine 0.10 20.0 206 15.0 Methionine 0.25 18.6 . 21A 15.1 lF value for interaction of treatment with steri— lized or unsterilized clay loam is not significant. The reason "Inia" wheat did not respond to amino acid infiltrations remains elusive, since the mature seed contains a large fraction of its nitrogen in arginine indicating the presence of enzymes necessary to metabolize it into usable substrates (Table 21). TABLE 21.-—Nitrogen distribution in total amino acids of oats and wheat seed. Amino Acids (us N/s dry wt) Oats Wheat "Rodney" "Ben Hur" "Ciano" "Genesee" "Inia" Aspartic Acid Threonine Serine Glutamic Acid Glycine Alanine Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Total 952 AA8 6AA 2,226 1,107 812 756 392 98 A91 92A 322 5A6 92A 756 2,520 13.918 516 329 6A0 2,905 821 566 538 A20 132 395 716 2A8 A07 575 650 1,6A1 11,A99 8A0 A90 952 2,6A6 1,218 826 728 A20 182 518 99A 36A 588 812 966 2,800 15.3““ 602 A06 616 2.870 966 71A 910 56 105 A3A 910 266 A62 532 588 1,A56 11,893 633 AA? 7A2 3,A10 1,109 808 1,109 2A 167 A9A 965 272 50A 69A 790 2.391 14,559 52 Amino Acid Infiltration: Utilization of Arginine During Early Seedling Development A major fraction of the nitrogen in the storage pro— tein of the seed used in the studies relating seed pro- tein content with subsequent seedling growth and yield was in arginine (Table 21). Therefore, enzymes necessary for its utilization in the synthesis of other amino acids and proteins at the growing axis should be present in the germinating seedling. In addition, if these enzymes are present they should function in the utilization of in- filtrated arginine. Six day-old etiolated wheat seedlings from seed infiltrated with 1.0 M arginine had a pronounced increase in total amino acids compared to untreated controls (Table 22). Similar increases were manifested in 9 and 10 day- old etiolated wheat seedlings and in 10 day-old seedlings grown in the light (Tables 22 and 23). Striking increases in aspartic acid were evident. Glutamic acid was higher in treatments than in controls of 6 and 10 day-old etio- lated seedlings and 10 day-old seedlings grown in the light. Aspartic and glutamic acid are the prime acceptors for ammonia, however, the resulting amides do not appear in the total amino acid analysis because of the hydrolysis procedure in preparing the sample for total amino acid analysis. Therefore, it is reasonable to conclude from these data that aspartate and to a lesser extent glutamate 53 TABLE 22.--Amino acid analysis of etiolated "Genesee" wheat seedlings from control and seed infiltrated with 1.0 M arginine. Age (days) Amino acids 6 9 (“MOleS/ Total Free Total Free g dry wt) Cont Arg Cont Arg Cont Arg Cont Arg Aspartic acid 397 5A8 7.A 7.5 265 317 6.6 8.0 Threonine A9 85 8.A l3.A A8 6A 7.9 7.2 Serine A7 93 9.0 7.1 A3 69 6.6 9.A Asparagine * * 277 297 * * 198 192 Glutamic acid 122 187 31.1 28.6 110 110 9.2 1A.1 Glycine 96 161 3.1 2.6 81 100 1.5 1.5 Alanine 93 161 8.0 6.5 8A 112 6.3 6.A Valine 87 1A9 2A.6 19.2 76 91 13.2 13.1 Cystine * 2 * * 6 7 * * Methionine 12 21 * * ll 13 * * Isoleucine 51 85 12.2 9.9 AA 50 6.A 5.1 Leucine 80 133 5.3 A.A 52 7A 1.9 2.1 Tyrosine 23 A2 2.2 1.8 22 25 1.3 1.7 Phenyl— alanine 35 61 2.0 1.7 32 37 1.5 1.A Lysine 35 A7 3.7 5.1 71 119 6.6 10.2 Histidine 17 16 9.1 6.9 27 3A 8.7 7.A Arginine 29 32 1.2 2.5 A5 52 A.5 3.2 Total 1,173 1,823 A0A AlA 1,017 1,27A 280 283 Total (mg N/s dry wt) 18.6 28.0 9.9 10.3 18.0 22.7 7.2 7.1 * Present in trace amounts or hydrolyzed in the case of asparagine. 5A TABLE 23.--Amino acid analysis of ten day-old "Genesee" wheat seedlings grown in the light and in the dark from control and seed infiltrated with 1.0 M arginine. Amino Light Dark (6331::/ Total Free Total Free g dry Wt) Cont Arg Cont Arg Cont Arg Cont Arg Aspartic acid 2A2 356 2.3 l3.A 5AA 638 10.1 12.2 Threonine 83 103 2.7 A.9 80 89 8.2 9.0 Serine 79 112 5.6 11.1 9A 91 10.6 13.2 Asparagine * * 98.2 230 * * 29A AAA Glutamic acid 191 21A 2A.3 A9.A 1A9 161 3A.6 3A.8 Glycine 161 198 A.A 12.6 137 1A6 1.7 2.3 Alanine 16A 210 A.9 15.1 119 132 5.9 8.2 Valine 135 182 5.5 9.1 130 133 8.7 12.8 Cystine 28 27 * * * * * * Methionine l 12 * * 18 25 * * Isoleucine 79 106 2.7 3.6 66 71 1.3 A.2 Leucine 138 203 1.9 2.8 108 128 1.5 1.9 Tyrosine A9 70 0.5 0.6 38 55 A.9 3.7 Phenyl- alanine 72 113 1.1 1.8 55 68 3.3 3.3 Lysine 156 158 1.7 1.9 155 128 7.6 9.0 Histidine A2 A3 1.7 1.3 A2 38 10.3 12.0 Arginine 128 127 1.A 2.6 120 105 8.3 9.3 Total 1,7A8 2,23A 159 360 1,855 2,008 All 580 Total (mg N/s dry wt) 33.6 A0.A 3.7 8.A 3A.A 35.A 10.6 15. * Present in trace amounts or hydrolyzed in the case of asparagine. 55 and their amides rise over controls in response to the arginine infiltrated into the seed. This was expected since aspartic and glutamic acid serve as intermediates in transferring nitrogen in storage proteins to amino acid residues used in the de novo synthesis of proteins at the growing axis. Amino acid analysis of 12 day-old etiolated seed- lings was indicative of the preceding results (Table 2A). However, the seedlings were beginning to senesce and die (rise in free amino acids) which probably accounted for the subtle differences in total amino acids, aspartic and glutamic acid as noted earlier. Total and free amino acid analysis of control and infiltrated wheat seed confirmed the increase in nitrogen content as determined by micro-Kjeldahl procedures (Table 2A). The increase in total nitrogen in treated seed was due to arginine infiltration. Relative differences in the free amino acid content of the seedlings were not large and in general corres- ponded to the differences in total amino acids. The total mg N/g dry weight, based on amino acid analysis, was higher in the seedlings than in the seed planted (Table 2A). This is misleading, but rectifiable. Consider the control of 10 day-old etiolated seedlings which contained 3A.A mg N/g dry weight. Each seedling 56 TABLE 2A.--Amino acid analysis of twelve day-old etiolated "Genesee" wheat seedlings and of seed from control and seed infiltrated with 1.0 M arginine. . 12 day-old Seed Amino (“agigi/ Total Free Total Free g dry Wt) Cont Arg Cont Arg Cont Arg Cont Arg Aspartic Acid A31 A52 5.A 9.1 A3 A2 0.7 0.A Threonine 55 58 9.8 9.7 29 28 0.1 0.5 Serine 58 60 11.8 13.5 AA 37 0.A 1.1 Asparagine * * 3A3 327 * * 2.7 0.9 Glutamic acid 118 128 28.0 28.6 205 197 * * Glycine 96 106 1.8 1.7 69 65 0.5 0.A Alanine 99 105 8.8 9.3 51 A6 0.6 1.0 Valine 85 9A 16.A 18.8 65 59 0.3 0.2 Cystine * * * * 2 l * * Methionine 15 17 * * 8 6 * * Isoleucine 38 A7 5.6 6.2 31 30 0.2 0.1 Leucine 7A 79 2.1 2.7 65 60 0.2 0.2 Tyrosine 25 28 3.5 3.8 l9 18 0.2 0.1 Phenyl- alanine 36 38 3.8 A.2 33 31 0.2 0.1 Lysine 78 85 22.8 22.9 19 27 * * Histidine 38 36 17.7 15.5 1A 18 0.1 0 l Arginine 56 52 10.6 1.2 26 1A2 * 1A3 Total 1,302 1,385 A91 A7A 723 807 6 1A8 Total (mg N/s dry wt) 22.7 23.8 12.9 12.0 11.9 18.2 0.1 8.1 * Present in trace amounts or hydrolyzed in the case of asparagine. 57 weighed approximately A mg, therefore, 0.138 mg N is con- tained per seedling. The seed planted weighed approxi- mately 38 mg, of which 0.A52 mg is N. Therefore, the seedlings do not contain more nitrogen than can be ac- counted for from the seed itself. CONCLUSIONS AND SUMMARY To a large extent, storage proteins serve as a source of nitrogen and amino acids which are utilized in the de novo synthesis of functional proteins at the grow- ing axis. It seems logical that high protein seeds would produce larger seedlings and higher yields than low pro- tein seeds of the same genotype. This relationship has been shown herein, with several wheat varieties and oats. The enhanced protein content of the seed was due to loca- tions or chemical and nitrogen applications. Seed weight, per cent moisture or nitrate content were not related to subsequent seedling growth or yield. Infiltrating wheat and oat seed with amino acids, urea and various amino acid, purine and pyrimidine pre- cursors produced varied results. In several experiments, wheat seed vacuum infiltrated for 3 hours in a 1.0 M arginine solution produced larger seedlings. In addition, oat seed infiltrated in a 0.67 M arginine solution or in a solution of 11 amino acids yielded more grain than controls in l of 2 field tests. Arginine is a major storage form of nitrogen in the wheat and oat seed used in these studies. The enzymes necessary for arginine metabolism are presumably present 39 in the germinating seed and would enable the utilization of infiltrated arginine. Amino acid analysis of etiolated seedlings during various stages of development, from un— treated and infiltrated seed, provides evidence for arginine metabolism. There is a corresponding rise in aspartic and glutamic acids and their amides which serve as central intermediates in the utilization of the guani— dino nitrogen of arginine. Further evidence that arginine serves as a nitrogen source was shown when foliar sprays of arginine produced similar effects of seedling growth and tiller production as urea sprays, which are known to supply ammonia. The amino acid data of etiolated seedlings and the enhanced seedling vigor of infiltrated seeds strongly suggests the internal localization of arginine at a site where it can be metabolized, rather than a peripheral accumulation on the seed coat. The effect of infiltrating arginine into seed on seedling growth and its utilization by etiolated seed- lings provides further evidence for the relationship of seed protein content with subsequent seedling growth and yield. Although the use of high protein seed to enhance future crop production is plausible, the technique of infiltrating arginine or other amino acids into seed may be of limited practical significance. However, the critical question which arises from these studies is: 60 can the storage protein of seed be altered genetically to contain high levels of basic amino acid residues (i.e. arginine) which can be metabolized upon germination and produce an effect throughout the life of the plant? LITERATURE CITED 61 10. LITERATURE CITED Albaum, H. G. 19A3. Relationship between nitrogen transport and metabolism in the oat seedling. Amer. J. Bot. 30:302—305. Albaum, H. G: and P. P. Cohen. 19A3. Transamination and protein synthesis in germinating oat seed- lings. J. Biol. Chem. 1A9:l9-27. Altschul, A. M. 1967. Food proteinsf New sources from seeds. Science. 158:221-226. Association of Official Agricultural Chemists. 1965. Official Methods of Analysis of the Associa- tion of Official Agricultural Chemists. Pub- lished by Association of Official Agricultural Chemists. Washington, D.C. 10th ed:7AA—7A5. Beevers, L. and W. E. Splittstoesser. 1968. Pro- tein and nucleic acid metabolism in germinating peas. J. Exptl. Bot. 19:698-711. Boulter, D. and J. T. Barber. 1963. Amino-acid metabolism in germinating seeds of Vicia faba L. in relation to their biology. New Phytol. 62:301-316. Brown, L. R. 1967. The world outlook for con- ventional agriculture. Science. 158:60A-611. Brown, R. T. 1967. Influence of naturally occur- ring compounds on germination and growth of Jack Pine. Ecology. A8:5A2-5A6. Chibnall, A. C. 1939. Protein Metabolism in the Plant. Yale University Press, New Haven, Conn. 306pp. Clark, B. E. and N. H. Peck. 1968. Relationship between the size and performance of snap bean seeds. N. Y. State Agr. Exp. Sta. Res. Bull. 8l9zA-30. ' 62 ll. 12. 13. l“. 15. 16. 17. 18. 19. 20. 63 Danielson, C. E. 1956. Plant proteins. Ann. Rev. Plant Physiol. 7:215—236. Daussant, J., N. J. Neucere and E. J. Conkerton. 1969. Immunochemical studies on Arachis hypogaea proteins with particular reference to the re— serve proteins. II. Protein modification during germination. Plant Physiol. AA:UBO—A84. Ferrari, A. 1960. Nitrogen determination by a con- tinuous digestion and analysis system. N. Y. Acad. Sci. 87:792-800. Finney, K. F., J. W. Meyer, F. W. Smith and H. C. Fryer. 1957. Effect of foliar spraying of Pawnee wheat with urea solutions on yield, protein content and protein quality. Agron. J. 49:3Al-347. Folkes, B. F. and E. W. Yemm. 1958. The respira- tion of barley plants. X. Respiration and metabolism of amino acids and proteins in germinating grain. New Phytol. 57:106-131. Galligar, G. C. 1938. Correlation between the growth of excised root tips and types of food stored in the seed. Plant Physiol. 13:599- 609. Goerlitz, H., H. Koriath, G. Rinno and G. Sprecht. 1967. Effects of nitrogen fertilization on content and yield of crude protein and other quality properties of harvested products. Deut. Landwirt. 18:168-172. (Chem. Abstr. 67:72890). Grace, N. H. 1939. Treatment of plant seed. Canada Patent 383,3“5, Aug. 15. (Chem. Abstr. 33:8350). Hamilton, P. B. 1963. Ion exchange chromatography of amino acids. A single column, high resolv- ing, fully automatic procedure. Anal. Chem. 35:2055-206“. Harris, G. P. 1956. Amino acids as sources of nitrogen for the growth of isolated oat embryos. New Phytol. 55:253-268. 21. 22. 23. 2A. 25. 26. 27. 28. 29. 30. 6A Heyl, G. E. 1937. Treating seeds with fertilizing and fungicidal substances. U. S. Patent 2,081,667, May 25, and U. 3. Patent 2,083,065, June 8. (Chem. Abstr. 3125096). Ingle, J. and R. H. Hageman. 1965. Metabolic changes associated with the germination of corn. II. Nucleic acid metabolism. Plant Physiol. “0:98-53. Jones, V. M. and D. Boulter. 1968. Arginine metabolism in germinating seeds of some mem- bers of the Leguminosae. New Phytol. 64: 925-93“. Joy, K. W. and B. F. Folkes. 1965. The uptake of amino acids and their incorporation into the proteins of excised barley embryos. J. Exptl. Bot. 16:6A6-666. Kasting, R. and C. C. Delwiche. 1958. Ornithine, citrulline and arginine metabolism in water- melon seedlings. Plant Physiol. 33:350—35A. Kaufmann, M. L. and A. A. Guitard. 1967. The effect of seed size on early plant develop- ment in barley. Can. J. Plant Sci. A7: 73-78. Kidd, F. and C. West. 1919. Physiological pre- determination: The influence of the physio— logical condition of the seed upon the course of subsequent growth and upon the yield. Ann. Applied Biol. 5:112-142, 157-170, 220-251. Kiesselback. T. A. 192“. Relation of seed size to yield of small grain crops. J. Amer. Soc. Agron. 16:670-682. Klein, S. and B. M. Pollock. 1968. Cell fine structure of developing lima bean seeds re- lated to seed desiccation. Amer. J. Bot. 55:658-672. Kloz, J., V. Turkova and E. Klozova. 1966. Pro- teins found during maturation and germination of seeds of Phaseolus vulgaris L. Biologia Plantarum (Praha). 8:I6A-l73. 31. 32. 33. 3M. 35. 36. 37. 39. U0. Al. 65 Larson, L. A. and H. Beevers. 1965. Amino acid metabolism in young pea seedlings. Plant Physiol. “0:42A-A32. 1968. The effect soaking pea seeds with or without seed coats has on seedling growth. Plant Physiol. u3z255-259. Lowe, R. H. and J. L. Hamilton. 1967. Rapid method for determination of nitrate in plants and soil extracts. J. Agr. Food Chem. 15:359-361. Lugg, J. W. H. and R. A. Weller. 1941. Protein metabolism in seed germination. Biochem. J. 35:1099-1105. Maretzki, A., L. G. Nickell and M. Thom. 1969. Arginine in growing cells of sugarcane. Nutritional effects, uptake and incorporation into proteins. Physiol. Plant. 22:827-839. Meiss, A. N. 1952. The formation of asparagine in etiolated seedlings of Lupinus albus L. Conn. (New Haven) Agr. Exp. Sta. Bull. 553:7“pp. Mertz, E. T., L. S. Bates and O. E. Nelson. 196U. Mutant gene that changes protein composition and increases lysine content of maize endo- sperm. Science. 145:279-280. Mihailescu, G., D. M. Cachita, N. Grosu and I. Ritu. 1968. Aminobenzoate esters stimulating plant growth and deveIOpment. Rom. 51:06U:2pp. (Chem. Abstr. 71:21111). Neatly, K. W. and A. G. McCalla. 1938. Correlation between yield and protein content of wheat and barley in relation to breeding. Can. J. Res. 16:1-15. Oaks, A. 1966. Transport of amino acids to the maize root. Plant Physiol. “1:173-180. Paul, K. B. and C. Nozzolillo. 1969. The uptake of phenylalanine during imbibition by seeds of the garden pea, Pisum sativum. Can. J. Bot. 97:1685-1691. M2. “3. AU. “5. U6. “7. AB. “9. 50. 51. 52. 66 Perry, T. L., D. Stedman and S. Hansen. 1968. A versatile lithium buffer elution system for single column automatic amino acid chroma- tography. J. Chromatog. 38:“60—“66. Pollock, B. 1969. Imbibition temperature sensi— tivity of lima bean seeds controlled by initial seed moisture. Plant Physiol. th907-911. Popov, A. l9u3. The germinating power of cereals in relation to the protein content of the grain. Angew. Botan. 25:150-165. (Chem. Abstr. 38:5536). Randal, J. 1967. FPC new hope for undernourished. BioScience. 17:257-258. Ries, S. K., H. Chmiel, D. R. Dilley and P. Filner. 1967. The increase in nitrate reductase activity and protein content of plants treated with simazine. Proc. Natl. Acad. Sci. U. S. 58:526—532. Ries, S. K., C. J. Schweizer and H. Chmiel. 1968. The increase in protein content and yield of simazine-treated crOps in Michigan and Costa Rica. BioScience. 18:205-208. Robertson, R. N., H. R. Highkin, J. Smydzuk and F. W. Went. 1962. The effect of environmental conditions on the development of pea seeds. Aust. J. Biol. Sci. 15:1-15. Schulze, E. 1898. Ueber den umsatz der eiweisstoffe in der lebenden pflanze. Z. Physiol. Chem. 2u:18—11u. (A. c. Chibnall, 1939). Shargool, P. D. and E. A. Cossins. 1969. Further studies of L-arginine biosynthesis in germi- nating pea seeds. Evidence for the presence of argininosuccinate synthetase in cotyledon extracts. Can. J. Biochem. h7zu67—A75. Sinha, R. N. 1969. Effect of presoaking seeds with plant-growth regulators and nutrient solution on dry matter production of rice. Madras Agr. J. 56:16-19. (Chem. Abstr. 71:48512). Splittstoesser, W. E. 1969. Metabolism of arginine by aging and 7 day old pumpkin seedlings. Plant Physiol. “4:361-366. 53. 5A. 55. 56. 57. 58. 59. 60. 61. 62. 63. 67 Splittstoesser, W. E. 1969. Arginine metabolism by pumpkin seedlings. Separation of plant extracts by ion exchange resins. Plant and Cell Physiol. 10:87-94. Stahamann, M. A. 1963. Plant proteins. Ann. Rev. Plant Physiol. 1A:137—158. Steiner, A. M. 1969. Influence of substrate re- serves on cell growth and cell-wall synthesis. Naturwissenschaften. 56:“23. Steward, F. C., R. F. Lyndon and J. T. Barber. 1965. Acrylamide gel electrOphoresis of soluble plant proteins: A Study on pea seedlings in relation to development. Amer. J. Bot. 52:155-16“. Stewart, C. R. and H. Beevers. 1967. Gluconeo- genesis from amino acids in germinating castor bean endosperm and its role in transport to the embryo. Plant Physiol. “221587-1595. Sure, B. and W. E. Tottingham. 1916. Relation of amide nitrogen to the nitrogen metabolism of the pea plant. J. Biol. Chem. 26:535-548. Terman, G. L., R. E. Ramig, A. F. Dreier and R. A. Olson. 1969. Yield—protein relationships in wheat grain, as affected by nitrogen and water. Agron. J. 61:755-759. Van Slyke, D. D. and A. J. Hiller. 1933. Determina- tion of ammonia in blood. J. Biol. Chem. 102:499-50“. Vickery, H. B. and G. W. Pucher. 19A3. Amide metabolism in etiolated seedlings. I. Asparagine_and glutamine formation in Lupinus angustifolius, Vicia atropurpurea, and Cucurbita pepo. J. Biol. Chem. 150:197-207. Villegas, E., C. E. McDonald and K. A. Gilles. 1968. Variability in the lysine content of wheat, rye, and triticale proteins. Inter— national Maize and Wheat Improvement Center, Mexico. Res. Bull. 10:1-32. Wang, D. 1968. Metabolism of amino acids and amides in germinating seeds. Contrib. Boyce Thompson Inst. 2u:109-115. 68 6A. Weissman, G. S. 1964. Effect of ammonium and nitrate nutrition on protein level and exudate composition. Plant Physiol. 39:9A7-952. 65. Wilson, D. G., K. W. King and R. H. Burris. 195A. Transamination reactions in plants. J. Biol. Chem. 208:863-87u. General References Altschul, A. M., L. Y Yatsu, R. L. Ory and E. M. Engleman. 1966. Seed proteins. Ann. Rev. Plant Physiol. 17:113-136. Bonner, J. and J. E. Varner. 1965. Plant Biochemistry. Academic Press, New York and London. 105App. Fowden, L. 196“. The chemistry and metabolism of recently isolated amino acids. Ann. Rev. Biochem. 33:173-204. Mahler, H. R. and E. H. Cordes. 1966. Biological Chemistry. Harper and Row, Publishers, New York and London. 872pp. Schroeder, W. A. 1968. The Primary Structure of Pro- teins. Harper and Row, Publishers, New York, Evanston and London. 210pp. Schultz, H. W. and A. F. Anglemeir. 196A. Symposium on Foods: Proteins and Their Reactions. The Avi Publishing Co., Inc., Westport, Conn. A72pp. APPENDIX 69 APPENDIX I Hoagland's solutionl Chemical Stock solution Final solution (g/l) (m1 stock/l H20) CaC12°2H20 1A7 1.5 K280“ 87 1.0 MgSOu°7H2O 98.6 2.5 KH2POu 27.2 2.5 Fe (Chelate 12%) 16.6 2.5 Minor elements: 1.25 ZnSOH'7H2O 0.088 H3808 1.1uu MnCl2-AH2O 0,720 CuSOu-5H20 0.032 H2MOu-H2O 0.008 KOH 56 adjust pH to 6.3 Equal quantities of 1.0 M stock solutions of KNO and Ca(NO3)2-UH2O for the following NO3 concentrations: 2 mM 3 mM U mM 8 mM 16 mM ml stock/l H2O: 0.66 0.99 1.33 2.66 5.32 1Modified from Hoagland, D. R. and D. I. Arnon. 1938. The water-culture method for growing plants without soil. Univ. Calif. Agri. Exp. Sta. Circ. 3A7. 70 MICIIWIWI[1177)[IIEJMWIWIWIVs