The phloem exudation technique using ethylenediaminetetraacetic acid (EDTA) was evaluated In studies of amino acid translocation in Pisum sativnm L. seedlngs. Exudation of phloem sap from cut petioles of fuily expanded leaves was enhanced by EDTA (20 millimolar disodium salt [pH 7.01). Amino acids (mainly asparagine, homoserine, glutamate, and also aspartate and serine) were present in petiole exudates from EDTA-treated leaves at levels which were commonly 5-to 10-fold (or more) higher compared with water-treated controls. Exudation was greater from darkened leaves, and the pattern of amino acids was markedly different from the more uniform mixture leaking from water-treated controls.After feeding 'C-labeled amino acids to the leaf blade, distribution of radioactivity in components of the exudate differed from that of the leaf tissue, suggesting selectivity of amino acid loading. 1'ClAsparagine was converted to 2-hydroxysuccinamic acid and to other amino acids by the leaf, but was recovered in exudate mainly as asparagine (60%) and aspartate (30%). SImilarly, in the exudate, 65 to 70% of the label from 114CI-aspartate was in amino acids, although in the leaf tissue 50% was in the organic acid fraction and only 11% remained as aspartate. Metabolism of asparagine and aspartate was essentially the same in intact leaf blades as in EDTA-treated leaves. Despite the possibility of EDTA damage in the petiole, phloem loading of amino acids appeared to be relatively unimpaired.Although the amount of labeled material appearing in the exudate is less than the amount translocated in the intact plant, the technique is useful in the study of amino acid transport.Mature, transpiring leaves import nitrogen from the roots, with varying proportions of nitrate and organic nitrogen, depending on species and growth conditions (17). In Pisum sativum, the nitrogen level of the growing leaves stabilizes when the leaves are fully expanded (3), thus most of the incoming nitrogen must be reexported. In a continuing study of amino acid metabolism and transport in peas (3), the redistribution of amino acids in phloem has been investigated.Several methods have been used in the past to obtain phloem samples from leaves. Phloem sap was collected from severed inflorescences of palms and some Agavacae (22) Phloem sap has been collected from the severed stylets of aphids (10) and amino acids were detected in aphid stylets exudate from willows (16,20) and from peas (2). The quantities of sap obtained are small and the difficulty in prepositioning the aphids limit the usefulness of this method in such studies.None of these techniques seemed suitable for collection of the phloem sap exported from fully expanded leaves of vegetative plants. Although some leaves do exude a phloem sap through the cut petiole (12), the quantities thus obtained are minute. King and Zeevaart (1 1) described an EDTA-promoted exudation of phloem sap from detached leaves. The method has subsequently been used with a variety of plants by other research groups (6,8,9,...

Amino acid metabolism and transport was investigated in the leaves of 3-week-old nonnodulated seedlings of Pisum sativum L. Xylem sap entering the shoot contained nitrate (about 5 millimolar), and amino compounds (11 millimolar) of which 70% was asparagine plus glutamine; aspartate and homoserine were also present. Mature leaves showed stable nitrogen levels and incoming nitrogen was redistributed to growing leaves. Younger leaves, still enclosed in the stipules, showed negligible rates of transpiration, suggesting that most of their nitrogen must arrive in the phloem."C-Labeled amides and amino acids were supplied to detached shoots through the xylem, and metabolism and redistribution were followed over 12 hours in light. Asparagine entering mature leaves was reexported directly to young leaves, with relatively little metabolic conversion. Substantial amounts of glutamine were converted to glutamate, which was exported (with unchanged amide) with little further conversion. The pattern of redistribution was confirmed when "C-labeled amino acids were applied directly to the under surface of mature leaves. Labeled compounds were found in the phloem exudate from treated leaves, and the composition resembled the pattern of labeling in the compounds arriving in the young developing leaves. from mature to developing leaves was not followed in detail, nor were labeled compounds recovered in the developing leaves identified. Export from the mature leaves of label from amino acids (applied as tracers to the leaf surface) has been demonstrated in several plants (1 1, 20, 22).In P. sativum seedlings, the xylem sap contains a mixture of nitrate and organic nitrogenous compounds, mainly asparagine and glutamine, but also homoserine, asparate, and a few other amino acids (4,25). Experiments with lWN-labeling showed that pea leaves utilize the nitrogen from nitrate and from asparagine and glutamine for further amino acid metabolism (5). In these pea seedlings, the young leaves of the vegetative apex are enclosed within the stipules of the next oldest, expanding leaves, and there is little transpirational flow to the apical leaves. Young pea leaves are therefore a useful system in which to study redistribution of nitrogen from the mature leaves.Uptake and redistribution of 14C-labeled amides, the major nitrogenous compounds in the xylem of pea (25), was followed in mature, expanding, and immature leaves of nonnodulated plants over 12 h in the light, after supply to the shoot in the transpiration stream. Movement of amino acids from mature leaves to immature leaves and other parts of the plant was investigated by application of 14C-labeled amino acids to the lower surface of mature leaves.Nitrogen required for growth of plant shoots is transported from the roots via the xylem as a mixture of nitrate and organic nitrogen, the proportions varying with species and conditions (17). Mature leaves (vigorously transpiring) receive most of this xylemborne nitrogen (8,19). Nitrogen levels stabilize in fully-expanded leaves (4) an...

In the young leaves of pea (Pisum sativum L.) plants, there was a diurnal variation in the levels of amino acids. In the light, total amino nitrogen increased for the first few hours, then stabilized; in the dark, there was a transient decrease foliowed by a gradual recovery. Asparagine, homoserine, alanine, and glutamine accounted for much of these changes. The incorporation of 15N into various components of the young leaves was foliowed after supply of 15N-nitrate. 15N appeared most rapidly in ammonia, due to reduction in the leaf, and this process took place predominantly in the light. A large proportion of the primary assimilation took place through the amide group of glutamine, which became labeled and turned over rapidly; labeling of glutamic acid and alanine was also rapid. Asparagine (amide group) soon became labeled and showed considerable turnover. Slower incorporation and turnover were found for aspartic acid, y-aminobutyric acid, and homoserine.Synthesis and turnover of afl of the amino acids continued at a low rate in the dark. y-Aminobutyric acid was the only compound found to label more rapidly in the dark than in the light.During much of the life of the plant, the developing leaves constitute the major sink for nitrogen transported from roots. The nitrogen supply consists of nitrate together with a varying proportion of organic nitrogen, depending on the species and conditions (22). The amides glutamine and asparagine are usually predominant as transport and storage forms of nitrogen (22). In field peas (Pisum arvense), over 50% of the nitrogen in bleeding xylem sap can be organic, with the amides and homoserine as major components (24).Glutamate dehydrogenase has been considered to play a major role in ammonia assimilation. It is present in many plant tissues and has been purified from pea roots (21); however, the enzyme has a low affinity for ammonia. Glutamine synthetase is present in plants, and recent work (7, 9, 19) indicates that it is located in chloroplasts; the affinity for ammonia may be up to 1,000-fold greater than that of glutamate dehydrogenase (20).Many reactions are known to involve glutamine, and the recent demonstration of the glutamate synthase reaction in plants (13) provides an attractive system both for utilization of transported glutamine, and (together with glutamine synthetase) for the primary assimilation of ammonia (review by Miflin, 17). However, the presence (or absence) of enzymes under assay conditions in extracts is not a sure indication of the role of the enzyme ' Supported by a grant from the National Research Council of Canada.2 Present address: Pflanzenphysiologisches Institut der Universitat, Altenbergrain 21, 3013 Bern, Switzerland.I To whom reprint requests should be addressed.within the plant, nor can the relative flow of metabolites through alternate pathways be judged from enzyme activities. Additional information on the flow of nitrogen through the pools of various components of primary nitrogen assimilation has been obtained from studies using...

Short term (2-hour) incorporation of nitrogen from nitrate, glutamine, or asparagine was studied by supplying them as unlabeled (14N) tracers to growing pea (Pisum sativum L.) leaves, which were previously labeled with 15N, and then following the elimination of 15N from various amino components of the tissue. Most components had active and inactive pools. Ammonia produced from nitrate was assimilated through the amide group of glutamine. When glutamine was supplied, its nitrogen was rapidly transferred to glutamic acid, asparagine, and other products, and there was some transfer to ammonia. Nitrogen from asparagine was widely distributed into ammonia and amino compounds. There was a rapid direct transfer to glutamine, which did not appear to involve free ammonia. Alanine nitrogen could be derived directly from aspargine, probably by transamination. Homoserine was synthesized in substantial amounts from all three nitrogen sources. Homoserine appears to derive nitrogen more readily from asparagine than from free aspartic acid. A large proportion of the pool of y-aminobutyric acid turned over, and was replenished with nitrogen from all three supplied sources.The central role of glutamine in primary nitrogen metabolism of higher plants has been demonstrated by several workers. The glutamine synthetase system is present in roots (8) and leaves (7,18,19), and a number of experiments with '5N have shown that glutamine is rapidly labeled in primary assimilation. In kinetic studies with 15N-nitrate (3) in young pea leaves, the amide group of glutamine had the highest rate of incorporation of ammonia, followed by glutamic acid and alanine; asparagine was extensively labeled, but at a slower rate which did not appear to be due to primary assimilation. Enzyme studies (11) suggest that the amide group of glutamine is the preferred donor which is transferred to aspartic acid for asparagine synthesis. In pea plants, asparagine is important in transport of nitrogen in the xylem (5), and is also stored in leaves, having one of the largest pools of the amino acids (10). Homoserine is also present in large amounts in pea plants (9, 10).There are relatively few data available on utilization or degradation of asparagine (11). Asparaginase has been suggested as providing the main pathway for asparagine utilization. In developing lupin seeds, asparagine was utilized as a nitrogen source, with the nitrogen appearing predominantly in ammonia, glutamine, and alanine, and asparaginase was very active in extracts of the tissue (2). 3 To whom reprint requests should be addressed.This paper describes experiments using l5N and '4N labeling of growing pea leaves, which allowed a comparison of the fate of nitrogen from nitrate, glutamine, and asparagine, during a period of 2 hr. Both amides supplied nitrogen to a range of compounds, including homoserine. Nitrogen from asparagine was extensively redistributed, and it was shown that asparagine could provide nitrogen for glutamine synthesis. MATERIALS AND METHODSThe objective was to fol...

Nitrogen harvest index has been positively correlated with grain nitrogen content in wheat (Johnson et al. 1968). In contrast, Cox et al. (1986) and Halloran and Ire (1979) found no correlation between these two parameters. Dalling et al. (1976) found an association between nitrogen harvest index and grain protein concentration in wheat while Desai and Bhatia (1978)