Aminotransfer from Alanine and Glutamate to Glycine and Serine during Photorespiration in Oat Leaves

Abstract

ABSTRACT"N-Labeled glutamate and alanine were used to examine the photorespiratory nitrogen metabolism in oat (A vena sativa L.) leaf slices. Glutamate and alanine supply amino groups for glycine formation during photorespiration. The nitrogen flux from alanine to glycine was estimated to be 3 times higher than that from glutamate. It is concluded from these results that alanine is a direct and important amino donor for photorespiratory glycine formation in oat leaves. The 'N labeling of serine was almost as high as that of glycine during the initial period of the labeling experiments. Thereafter, the ratio of "N label in serine to "N label in glycine declined substantially.During photorespiration glycine is formed from glyoxylate. This process occurs in the leaf peroxisomes. The glycine enters the mitochondria, where two glycine are converted into one serine (24). In this complex reaction, CO2 and NH3 are liberated. The serine reenters the leaf peroxisomes, where serine:glyoxylate aminotransferase mediates the formation of hydroxypyruvate and glycine. By this process, one amino-nitrogen is directly recycled for glycine formation. The nitrogen of the other amino group, liberated as NH3, is refixed to glutamate via the glutamine synthetase/glutamate synthase pathway (1 1, 13, 22, 30). The finding that leaf peroxisomes contain glutamate:glyoxylate aminotransferase leads to the suggestion that the additional amino nitrogen needed for the formation of the second glycine is provided by this glutamate (13). However, there are a number of reports indicating that leaf extracts, or leaf peroxisomes, have substantial alanine:glyoxylate aminotransferase activity that may even exceed the glutamate:glyoxylate aminotransferase activity (4,7,21,24,25,28). Alanine is the main substrate for the formation of glycine also in the microbodies (peroxisomes) of the alga Mougeotia (29). In higher plants, most of the alanine:glyoxylate aminotransferase activity is attributed to the glutamate:glyoxylate aminotransferase (21,24,25). Glutamate:glyoxylate aminotransferase is also responsible for the glutamate:pyruvate and the alanine:a-ketoglutarate aminotransferase activities of leaf peroxisomes (3, 12,19,20 Preparation of Leaf Slices. The leaves were cut into slices (1.5-2.0 mm) with a razor blade. Four g of the leaf slices were vacuuminfiltrated in 40 ml of an osmotic medium consisting of 0.3 M sorbitol, 2 nim KH2PO4, 2 mM MgCl2, 1 mim MnCl2, 1 mm EDTA, and 5 mm Hepes/KOH, pH 7.9. Care was taken to release the vacuum slowly. Before use, the leaf slices were rinsed in a sieve with 40 ml of the same medium.Labeling Experiments. The leaf slices were transferred into a cuvette (8 x 5 x 2 cm) filled with 55 ml of the osmotic medium described above. The latter was bubbled with air for 3 h before being used. The leaf slices were stirred gently from the top, and the cuvette was illuminated with slide projectors from both sides the leaf slices were quickly transferred into a sieve, rinsed with 30 ml of water, and dipped several times into a large...

Glyoxylate Transamination in Intact Leaf Peroxisomes

“…This suggestion is quite different from the interpretation of in vivo feeding experimental results, from which alanine was suggested to participate 3 times more than glutamate in glyoxylate transamination in oat leaves (1). In order to have a 3-time preference ofalanine over glutamate in peroxisomal glyoxylate transamination, our results suggest that the relative concentration of alanine to glutamate should also be about 3 times in the cytosol.…”

“…This seems to be rather unlikely, although it is possible. Alternatively, the observation that in vivo alanine participated 3 times more than glutamate in glyoxylate transamination (1) may be due to the glutamate pool being larger than the alanine pool (Table III); this pool size effect was suggested not to be the case (1). Other possible reasons for the discrepancy include a difference in the oat varieties and a variation in the growth conditions under which the relative concentration of glutamate to alanine may alter.…”

“…Thus, a more appropriate approach to explore the relative contribution of the amino acids in glycine production is to observe the conversion of glycolate to glycine in intact peroxisomes in the presence of a combination of amino acids at concentrations expected to occur in vivo. This approach would also avoid the uncertainties of uptake and pool size ofdifferent amino acids in an in vivo study, as in the study with oat leaves (1) in which the workers attempted to eliminate these uncertainties.…”

Glutamine Synthetase-Glutamate Synthase Pathway and Glutamate Dehydrogenase Play Distinct Roles in the Sink-Source Nitrogen Cycle in Tobacco

“…The 15 N-labeling patterns of Glu, Gly, Ser, Ala, and Pro in this study correlate with the open flux of nitrogen by import and export of amino acids into and from the photorespiratory nitrogen cycle, respectively (Betsche, 1983). The synthesis of [2-15 N]Glu and the [2-15 N]Gluderived amino acids was inhibited by AZA in young (Somerville and Ogren, 1980;Blackwell et al, 1988;Ferrario-Méry et al, 2002).…”

Glutamate:Glyoxylate Aminotransferase Modulates Amino Acid Content during Photorespiration

“…Both Glu and Ala are used as amine donors for transamination to glyoxylate in the GGAT reaction. Estimates based on whole-leaf labeling analysis suggested that Ala contributed three times more amino groups to photorespiratory Gly formation than Glu did (Betsche, 1983), whereas another study based on feeding metabolites to purified peroxisomes concluded that Glu contributed more to glyoxylate transamination than Ala did (Yu et al, 1984). The cooperative change of Glu and Ala in the GGAT1 knockout line and the GGAT1 overexpression line suggests that both amino acids contribute to transamination for Gly formation.…”