Effect of Ammonium and Nitrate Nutrition on Protein Level and Exudate Composition

Abstract

Research efforts of recent years on the protein metabolism of plants have been primarily directed toward clarification of the cellular mechanism of protein synthesis. Less attention has been devoted to the analysis of those factors that determine protein concentration levels in organs of whole plants. A review on this subject is included in the monograph of Web-ster (18). Past efforts of the present investigator in this area have been concerned with the influence of the ammonium: nitrate ratio of the culture solution on protein level in shoots of young wheat seedlings grown in darkness (19, 20). The shoot protein level of plants provided with a simultaneous supply of ammonium and nitrate was found to be greater than that of plants grown with either ammonium or nitrate alone. The present study concerns itself with the effect of am-moninum and nitrate on protein synthesis in young leaves of sunflower grown in the light. Preliminary experiments indicated that protein levels were again at a maximum under ammonium plus nitrate conditions. Among the factors that may play a role in the determination of leaf protein level, one is the nature of the nitrogenous substances transferred to the leaves from the root system. In an attempt to explain the decline in protein experienced by detached leaves, Chibnall (5) suggested that roots provide some factor or factors necessary for leaves to maintain their protein levels. This hypothesis is relevant to the problem of protein level in attached leaves under investigation in this laboratory. It is possible that the simultaneous presence of ammonium and nitrate in the culture solution may lead to the synthesis in the roots of a complex of nitrogenous compounds which, when transported to the leaves, permits the establishment of a high protein level. Accordingly, a study was made of the amino acids, amides, and inorganic nitrogen ions in the exudate of decapitated sunflower plants that had been grown under ammonium, ammonium plus nitrate, and nitrate conditions. Materials and Methods Seeds of Helianthus annuus L. var. Mammoth Russian were soaked in distilled water for 1 hour and individually planted in paraffined cups containing 'Revised manuscript received July 9, 1964. 2 This investigation was conducted with the aid of Research Grant 200 provided by the Research Council of Rutgers University. washed sand. The cups were supported in 400 ml beakers which served as collectors of culture solution applied to the seedlings. Excess solution dripped through glass wool covered perforations in the bottom of the cups. Distilled water was added to the seedlings during the first 6 days of growth and this was followed by the application of a previously described culture solution lacking nitrogen (20) for a period of 4 days. On the morning of the eleventh day the plants were divided into 3 classes of 12 plants each and, after the removal of the cotyledons, 25 ml of complete culture solution containing nitrogen as either 0.01 M NH4CI, 0.005 M NH4Cl plus 0.005 M KNO,, or 0.01 M KNO% were supplied to the plants of the respective classes. A similar addition of complete culture solution was made the morning of the twelfth day and the following morning, after receiving their third allotment of complete culture solution, the plants were sliced in the hypocotyl region about 1 cm above sand level. Exudates were collected over a 1-hour period with fine-tipped 10 ul graduated pipettes and were applied directly to chromatography paper for amino acid and amide analysis. The complete procedure was repeated 4 times and each time the 2 sets of opposite leaves that had developed were collected, separated into lots of older and younger leaves, and dried in a forced draft oven at 75°. The dry tissue of each trial was weighed and ground, pooled with the tissue collected in other trials, and saved for analysis of total and protein nitrogen by Kjeldahl methods previously described (19). In a fifth repetition of this experiment the exudate was subjected to analysis of its ammonia and nitrate content. Ammonia was determined by the vacuum distillation method of Archi-bald (1) and nitrate was determined colorimetrically with phenoldisulfonic acid (9). In a second experiment, 2 groups of plants were grown in order to determine the effect of a single application of nitrogen on the nitrogenous composition of the exudate. Plants were grown in the usual manner for a period of 10 days, and the next morning, after removal of the cotyledons, each group of plants was divided into 4 classes. The first class received culture solution lacking nitrogen, and the other 3 received solutions containing ammonium, ammonium plus nitrate, or nitrate. The plants were decapitated and exudate was collected for 1 hour. Exudates from 1 group were used in the determination of amino acids 947