Amino Acid Metabolism of Pea Leaves

Abstract

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...

An Appraisal on the Source-to-Sink Relationship in Plants: an Application of Desorption Electrospray Ionization Mass Spectrometry Imaging

“…Asparagine is transferred to young leaves directly from roots and mature leaves by the xylem and phloem, respectively. 36 The results displayed in Figure 2e fully confirm this assumption: although asparagine is detectable in the three peppermint leaves, its presence is much more notable in the young leaf. Furthermore, note that asparagine is more concentrated at the edges of the mature leaf, which seems to indicate that these are the storage sites for this amino acid.…”

“…That is because growing leaves demand a huge amount of protein for growing. 36,37 Table 1 displays the amino acids and ureides detected in the peppermint leaves, for which appropriate and discernible images were generated upon DESI-MSI application. Figure 2 shows the chemical images for each of these identified amino acids/ureides, generated directly from the imprintings of the peppermint leaves at the three distinct development stages.…”

Asparagine Synthesis in Pea Leaves, and the Occurrence of an Asparagine Synthetase Inhibitor

“…Although the level of asparagine decreases in mature pea leaves, presumably due to selective export (20), labeling data suggests that pea leaf tissue is capable of asparagine synthesis (2). This has been confirmed here by analysis of detached shoots; aspartate can be shown to be an asparagine precursor if a transaminase inhibitor is used to prevent the rapid equilibration of aspartate carbon with the organic acid pool.…”

“…Asparagine synthetase (EC 6.4.5.4), catalyzing the glutamine-dependent amidation of aspartate, is active in germinating seedlings (9,15,17), but there is some question about the synthesis of asparagine in older, nongerminating tissue. '5N-Labeling patterns in pea leaves (2,3) and spinach (21) are consistent with the operation of asparagine synthetase, yet there are some difficulties in the acceptance of this pathway. '4C-Labeled aspartate is a poor precursor for asparagine in pea roots (13) and soybean cotyledons (17).…”

Gas Chromatography-Mass Spectrometry of N- Heptafluorobutyryl Isobutyl Esters of Amino Acids in the Analysis of the Kinetics of [15N]H4+ Assimilation in Lemna minor L

“…Questions remain concerning the relative importance of primary ammonia-assimilation pathways (24), the pathways of utilization of the various N sources arriving in the growing leaf (2), and the mechanisms and significance of amino acid accumulation in response to environmental stress (31). One a,pproach to these questions is through examination of the use of N-labeled precursors (1,2,4,8,12,24,28,29,32,33). However, 15N labeling studies with plant tissues are hindered by the lack of sensitivity inherent in traditional MS and optical emission spectrometry methods (6,9,13,14,34) and by the complexities of the endogenous pools of amino acids which lead to problems in the interpretation of isotopic labeling kinetics in the absence of mathematical models (24,29).…”