The Relationship between Nh3 Accumulation and Photorespiratory Activity

Additions of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase (GS), result in an increase in NH13 in seedling leaves of Cs (wheat I Triticum aestiwn cv. Kolibril and barley [Hordeum vulgare var Perthi) and C4 (corn IZea mays W6A x W182E1 and sorghum [Sorghun Vulgare var MK3001) plants. NH13 accumulation is higher in C3 (about 17.8 micromoles per gram fresh weight per hour) than in C4 (about 4.7 micromoles) leaves. Under ideal conditions, when photosynthesis is not yet inhibited by the accumulation of NH3, the rate of NH.1 accumulation is about 16% of the apparent rate of photosynthesis. A maximum accumulation of NH13 was elicited by 2.5 mlllmolar MSX and was essentially independent of the addition of N03during either the growth or experimental period. When 02 levels in the air were reduced to 2%, MSX resulted in some accumulation of NH,3 (6.0 micromoles per gram fresh weight per hour). At these levels of NH3, there was no significant inhibition of rates of CO2 fixation. There was also a minor, but significant, accumulation of NH,, in corn roots treated with MSX. Inhibitors of photorespiration (isonicotinic hydrazide, 70 milimolar, 2-pyridylhydroxymethanesulfonic acid, 20 mlhlmoar) or transaminase reactions (aminooxyacetate, 1 millmolar) inhibited the accumulation of NH. in both Cs and C4 leaves. Tbese results support the hypothesis that GS is important in the assimilation of NH3 in leaves and that the glycine-serine conversion is a major source of that NH1. In 1974, Mahon et al (9) suggested that the major reaction leading to the release of CO2 in photorespiration was the conversion of glycine to serine. If this were true, the release of NH3 by photorespiration would be as significant as the release of CO2. In C3 plants, the rates of photorespiration can be considerable, up to 25% the rate of photosynthesis (9). Thus, in a system where NH3 accumulation can be measured, NH3 derived from photorespiration could be far in excess of the NH3 produced by the reduction of N03 . Under normal conditions, however, little NH3 is recovered from leaf tissue (1, 21). Miflin and Lea (1 1) suggested that an active GS3 could be responsible for the efficient assimilation of ' Supported by the Natural Sciences and Engineering Research Council of Canada (Grant No. 2818), and to F. M. by grants from the FranceCanada Exchange Cooperation and the French Ministry of Foreign Affairs. 2Author to whom reprint requests should be addressed. 3Abbreviations: GS, glutamine synthetase; MSX, methionine sulfoximine; GOGAT, glutamate synthase; INH, isonicotinic hydrazide; aHPMS, 2-pyridylhydroxymethanesulfonic acid; AOA, aminooxyacetate; GDH, glutamate dehydrogenase. NH3, and subsequently additions of MSX, an inhibitor of GS, were shown to result in accumulations of NH3 (4, 5, 10, 12, 18) and in the inhibition of photosynthesis (13, 14). In 1978, Keys et al. (7) proposed a photorespiratory nitrogen cycle whereby NH3 released from glycine would be efficiently reassimilated by GS in the cytosol. The resultant glutamine (20) would be transferred to the chloroplast where it would serve as a substrate for GOGAT, and the resultant glutamate would serve as the N-donor in the transaminase reaction leading to glycine formation in the peroxisome. The overall reactions and potential sites of inhibition are illustrated in Figure 1. Sommerville and Ogren (16) showed that Arabidopsis mutants lacking GOGAT accumulated NH3 under conditions which permitted photorespiration. In mutants lacking serinetranshydroxymethylase activity, NH3 was necessary for the continued synthesis of glycine, again under conditions which permitted photorespiration (17). Thus, their results support the hypothesis of Keys et al (7). Recently, Platt and co-workers (13, 14, and personal communication) observed an accumulation of NH3 in spinach leaf discs and an inhibition of photosynthetic activity in the presence of MSX. Under conditions where photosynthesis was inhibited, they still saw an accumulation of NH3 in experiments where the 02 content was reduced to 2%. These conditions should have inhibited photorespiration (15, 19) and hence NH3 accumulation. These observations agree with neither the Arabidopsis-mutant studies (16, 17) nor with the original hypothesis of Keys et al (7, 20) as it suggests alternate and significant sources of NH3 in leaf tissue. Explanations for this apparent contradiction could be the excessively high levels of MSX (8 mM) used in Platt's experiments and to the use of leaf discs rather than whole leaves. In view of the fact that corn leaf pieces showed an accumulation of NH3 in the presence of MSX (12) and that high levels ofMSX (2.5 mM) caused a general inhibition of root metabolism (A. Oaks, unpublished), it seemed to us that the role of MSX in leaves needed to be reexamined. In this paper, we show that a maximum level of NH3 is released by relatively low concentrations of MSX (0.60-2.5 mM) and that this release is inhibited by low concentrations of 02, by standard inhibitors of photorespiration (INH, HPMS) and by a transaminase inhibitor (AOA) in leaves of both C3 and C4 plants. Under ideal conditions, when photosynthesis is not inhibited, the levels of NH3 accumulated in the presence of MSX are 3 to 4 times higher in C3 than in C4 leaves. MATERIALS AND METHODS Plant Matenal. Corn caryopsis (Zea mays W6A x W182E, supplied by the Wisconsin Seed Foundation, Madison, WI), wheat (Triticum aestivum cv Kolibri, supplied through the OECD program), barley (Hordeum vulgare var Perth, supplied by the Crop 177 www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 1983 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 71, 1983 0 0 AwrC)OH * ArC AOA