Assimilation of C 14 , N 15 labeled urea by excised apple & peach leaves

Abstract

The widespread use of urea as a nutrient spray is a reflection of its capacity to be absorbed and metabo-lized by the foilage of many plant species. Certain fruit crops, such as the peach, however, do not efficiently utilize nitrogen from foliar-applied urea (3, 7). It is not known whether the problem is one of absorption or subsequent metabolism of the applied urea. Experiments using urea-C14 (23) indicated assimilation of the carbon portion of urea but gave no conclusive evidence for assimilation of urea nitrogen. Evidence supporting assimilation of urea nitrogen in apple (2), citrus (11), coffee, cacao, and banana (4) is largely based on quantitative differences in leaf amino acid concentration following foliar application of urea. Such studies do not distinguish between the nitrogen derived from urea or that derived from proteolysis and consequently may not be unequivocal evidence for nitrogen absorption. This distinction can be made by incorporating the N15 isotope into the urea supplied. In view of the gross difference in utilization of foliar-applied urea, this study was conducted utilizing C14,N15 labeled urea to compare assimilation by apple and peach leaves. MATERIALS & METHODS To circumvent difficulties resulting from differential cuticular absorption, C14,N'5 labeled urea was supplied through the petiole. Fifty young peach [Prunus persica (L.) Batsch, var. Elberta] and apple (Pyrus Malus L. var. Delicious) leaves were placed individually in 2 ml of 0.05 M phosphate buffer containing 3 mg urea (98.7 % N'5 and 20Ac urea C14 specific activity 1 Ac//Amole) per ml. The leaves were illuminated for 20 hours with fluorescent and incandescent lights after which they were quick frozen and stored at-18 C until analyzed. Peach and apple leaves and watermelon (Citrullus vulgaris Schrad) seedlings also received urea C14 or urea N15 during a 12 hour absorption period in preliminary experiments. The frozen leaves were extracted with 80 % (v/v) ethanol. A protein hydrolyzate of the ethanol insoluble residue was prepared according to Block et al I (1). The ethanol extract was reduced in vacuo at 40 C and extracted with diethyl ether. The aqueous phase was purified for chromatography by ion exchange according to the method of Plaisted (15). The purified extracts were subjected to two dimensional descending paper chromatography, employing phenol-water (4: 1) and n-butanol-propionic acid-water (125:59: 83) on 18 X 22 inch sheets of Whatman No. 1 filter paper. The amino acids were located by dipping the dry chromatograms in 0.25 % (w/v) ninhydrin in acetone containing 5 % (v/v) glacial acetic acid and heating for 30 minutes at 60 C in an ethanol atmosphere. Radioautograms were prepared from the paper chromatograms in the usual manner. The amino acids of the purified extracts were also separated by ion exchange column chromatography by modifying the procedure of Moore et al. (13). One-half milliliter of each fraction was plated and counted for C14 on aluminum planchets. The remaining one-half ml of each fraction was analyzed for amino acids by the photometric ninhydrin procedure of Rosen (17). An aliquot from the planchet corresponding to a ninhydrin and/or radioactivity peak was used to verify the identity and purity of the amino acids by one dimensional paper chromatography employing the solvent systems described above. Nitrogen in the fractionated amino acid samples and in samples obtained during preparation and purification of the leaf sample was analyzed for N'5 in the mass spectrometer of the Stable Isotopes Laboratory of North Carolina State College. RESULTS A preliminary experiment was performed to determine the quantity of leaf material and urea-N'5 required to measure incorporation into the free amino acids. After a 12 hour absorption period, with 12 mg of urea-N'5 supplied to ten apple leaves through the petiole, N15 was incorporated into all of the amino acids analyzed (table I). The greatest enrichment was found in glutamic acid plus glutamine and ala-nine. Valine was also highly enriched, but contained an unidentified contaminant. The quantity of alpha amino nitrogen present in ten leaves was insufficient to accurately determine N15 incorporation into the minor acids. Absorption of the C'14,N'5 urea treating solution into apple leaves was essentially complete within 3 hours. Characteristic symptoms of urea injury ap-757