Ammonium accumulation is associated with senescence of rice leaves
Shu Jiuan Chen, Kuo Tung Hung & Ching Huei Kao
Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China (
author for correspondence)
Received 24 December 1996; accepted in revised form 21 February 1997
Key words: abscisic acid, ammonium, benzyladenine, leaf senescence, methionine sulfoximine, Oryza sativa
Abstract
The relationship between ammonium accumulation and senescence of detached rice leaves was investigated. Ammonium accumulation in detached rice leaves coincided closely with dark-induced senescence. Exogenous NH4Cl and methionine sulfoximine, which caused an accumulation of ammonium in detached rice leaves, promoted
senescence. Treatments such as light and benzyladenine, which retarded senescence, decreased ammonium level in detached rice leaves. Abscisic acid, which promoted senescence, increased ammonium level in detached rice leaves. The current results suggest that ammonium accumulation may be involved in regulating senescence. Evidence was presented to show that ammonium accumulated in detached rice leaves increases tissue sensitivity to ethylene. The accumulation of ammonium in detached rice leaves during dark-induced senescence is attributed to a decrease in glutamine synthetase activity and an increase in reduction of nitrate.
Abbreviations: ABA = abscisic acid; BA = benzyladenine; GOGAT = glutamate synthase; GS = glutamine
synthetase; MDA = malondialdehyde; MSO = methionine sulfoximine; STS = silver thiosulfate
1. Introduction
Leaf senescence is an integral part of leaf develop-ment. It is, like other development processes, a well organized and regulated process rather than a passive decay of the leaves. Ammonium assimilation changes considerably with the onset of senescence [4]. Gluta-mine synthetase (GS, EC 6.3.1.2), the key enzyme in the generally recognized GS/GOGAT pathway, plays a crucial role in the assimilation of ammonium [14]. GS activity is known to decrease during either natural or dark-induced senescence of leaves [12, 18, 21, 24–26, 29]. Decline in GS activity in leaves during senes-cence may result, at least in part, in an accumulation of ammonium in leaves. In fact, various investigations have been able to show the accumulation of ammoni-um in leaves during senescence [21, 29]. Ammoniammoni-um is considered to be toxic to plant cells [5]. Lauriere and Daussant [13] suggested that ammonium accumulation might be a factor contributing to senescence. However, no direct evidence has been provided to prove this
sug-gestion. Ammonium has been demonstrated to accu-mulate after the onset of senescence of detached wheat leaves [18, 29]. Thus, emphasis in the present inves-tigation was on the relationship between ammonium accumulation and senescence of detached rice leaves.
2. Materials and methods
Rice (Oryza sativa cv. Taichung Native 1) was cul-tured as previously described [10]. The apical 3-cm segments excised from the third leaves of 12-d-old seedlings were used. A group of 10 segments was floated in a Petri dish containing 10 mL of test solu-tions. Incubation was carried out at 27
C in darkness or in the light (40mol m
2s 1).
For protein determination, leaf segments were homogenized in 50 mm sodium phosphate buffer (pH 6.8). The extracts were centrifuged at 17,600 g for 20 min, and the supernatant liquids were used for determination of protein by the method of Bradford [3].
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Protein level was expressed as mg 1g 1fresh weight. For ammonium determination, leaf segments were homogenized in 0.3 mM sulphuric aicd (pH 3.5). The homogenate was centrifuged for 10 min at 39,000 g and the supernatant was used for determination of ammo-nium by the method described previously [15]. Ammo-nium level was expressed asmol g
1 fresh weight.
For nitrate determination, leaf segments were homoge-nized with double distilled water. The homogenate was centrifuged for 25 min at 17,600 g. The supernatant was used for determination of nitrate by the method described by Hecht and Mohr [7]. Nitrate level was expressed asmol g
1fresh weight.
MDA was extracted with 5% (w/v) trichloroacetic acid and determined according to Heath and Packer [6]. MDA is routinely used as an index of lipid peroxidation and was expressed as nmol g 1fresh weight.
For extraction of GS, leaf segments were homog-enized with 10 mM Tris-HCl buffer (pH 7.6, con-taining 1 mM mgCl2, 1mM EDTA and 10 mM 2-mercaptoethanol) in a chilled pestle and mortar. The homogenate was centrifuged at 15,000 g for 30 min and the resulting supernatant was used for determina-tion of GS activity. The whole extracdetermina-tion procedure was carried out at 4
C. GS was assayed by the method of Oak et al. [17]. One unit of GS activity is defined as 1mol L-glutamate-r-monohydroxamate formed per
min.
A stock solution of STS was prepared by mixing equal volumes of 0.01 M AgNO3and 0.04 M Na2S2O3
[16].
All experiments were repeated three times. Similar results and identical trends were obtained each time. The data reported here are from a single experiment.
3. Results
The senescence of detached rice leaves is characterized by a decrease in chlorophyll and protein levels [10]. The loss of chlorophyll and protein has been the princi-pal criterion of leaf senescence for the largest number of workers [28]. Since protein loss in detached rice leaves is prior to chlorophyll loss [10], the senescence of detached rice leaves was monitored by the decrease of protein in the present study. Figure 1 shows the time courses of protein, ammonium, and nitrate levels and GS activity in detached rice leaves incubated in dark-ness. A decrease in protein level in detached leaves was evident 2 days after leaf detachment. Ammoni-um level in control leaves remained unchanged during
Figure 1. Protein, ammonium, and nitrate levels and GS activity in
detached rice leaves during dark-induced senescence. Vertical bars represent standard errors (n=4).
the first day of dark incubation and increased subse-quently. The increase in ammonium level coincided closely with the decrease in nitrate level and GS activ-ity. Our results indicate that ammonium accumulation parallels the senescence of rice leaves under dark con-dition. Peeters and Van Laere [18] and Thomas [29] reported that ammonium accumulated after onset of leaf senescence.
It is clear from Figure 2 that light was effective in retarding senescence of detached rice leaves. Light treatment resulted in a lower ammonium level and a higher GS activity in detached rice leaves when com-pared with the dark control.
If ammonium accumulation plays a regulatory role in dark-induced senescence of detached rice leaves, it is expected that treatment of NH4Cl would increase
endogenous ammonium level and consequently pro-mote senescence. As indicated in Figure 3, this is
Figure 2. Influence of light on protein and ammonium levels and
GS activity in detached rice leaves. All measurements were made 3 days after treatment. Vertical bars represent standard errors (n=4).
indeed the case. It is also clear from Figure 3 that the increase in ammonium level in NH4Cl-treated
detached rice leaves was associated with the decrease in GS activity (Figure 3).
MSO is a structural analogue of glutamate, and serves as an irreversible inhibitor of GS [22, 23]. There is evidence that addition of MSO results in an accumu-lation of ammonium [1, 19, 20]. To characterize fur-ther the role of ammonium accumulation in regulating dark-induced senescence, detached rice leaves were
Figure 3. Influence of NH4Cl on protein and ammonium levels and GS activity in detached rice leaves. All measurements were made 3 days after treatment in the dark. Vertical bars represent standard errors (n=4).
incubated in the presence of various concentrations of MSO. As indicated in Figure 4, MSO decreased GS activity, increased endogenous ammonium level, and promoted senescence.
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Figure 4. Influence of MSO on protein and ammonium levels and
GS activity in detached rice leaves. All measurements were made 3 days after treatment in the dark. Vertical bars represent standard errors (n=4).
It has long been recognized that cytokinins are effective in retarding the senescence of most if not all leaves. The effect of cytokinins in retarding senescence is species- or variety-specific; for the rice variety used in this investigation, BA has been found to be the most
active cytokinin in retarding senescence in darkness [9]. The effect of BA on leaf senescence in relation to ammonium level and GS activity is presented in Figure 5. BA effectively retarded senescence, reduced ammonium accumulation, and increased GS activity in detached rice leaves. Among the known promotors of senescence, ABA has been studied most extensively. Figure 6 shows the effects of ABA on senescence, ammonium level and GS activity in detached rice leaves. ABA promoted senescence, increased ammo-nium accumulation, and decreased GS activity.
Since accumulation of ammonium coincides with the decrease in nitrate level during dark-induced senes-cence (Figure 1), it is of interest to know whether the changes in ammonium level in BA- and ABA-treated detached rice leaves are associated with those in nitrate level. Figure 7 shows that BA treatment had higher nitrate level, whereas ABA treatment had lower nitrate level than the control leaves. These results indi-cate that nitrate reduction is significant as a source of ammonium.
Lipid peroxidation is considered to be an important mechanism of leaf senescence [27]. Thus we examined the changes in the level of MDA during dark incuba-tion of detached rice leaves and the influence of NH4Cl
on MDA level in detached rice leaves (Figure 8). No increase in MDA level was observed in detached rice leaves during senescence. We also observed that the increase in MDA level did not parallel the concentra-tions of NH4Cl applied. These results seem to exclude
the possibility that ammonium-promoted senescence is mediated through lipid peroxidation.
Previously, we demonstrated that senescence of detached rice leaves is mediated through an increase of tissue sensitivity to ethylene [11]. It is most likely that NH4Cl or MSO may change ethylene sensitivity
of detached rice leaves. This possibility was tested by using ethylene action inhibitor, Ag+
[2]. In the present study, STS was used as a source of Ag+
. The effect of STS on NH4Cl- and MSO-promoted senescence of
detached rice leaves is shown in Figure 9. STS was observed to inhibit NH4Cl- and MSO-promoted
senes-cence of detached rice leaves.
4. Discussion
The present study shows that ammonium accumulates in detached rice leaves during dark-induced senes-cence. In the photorespiration, the glycine decarboxy-lase reaction produces not only CO2but also an
equiv-Figure 5. Changes in protein and ammonium levels and GS activity
in detached rice leaves treated with BA. Detached rice leaves were treated with either water or 10M BA in the dark. Vertical bars represent standard errors (n=4).
alent quantity of ammonium. Since our experiments were conducted in darkness, ammonium is unlikely to have been produced from photorespiration. The assim-ilation of ammonium requires carbon skeltons, energy and GS. We have observed that GS activity in detached rice leaves decreased during dark-induced senescence.
Figure 6. Changes in protein and ammonium levels and GS activity
in detached rice leaves treated with ABA. Detached rice leaves were treated with either water or 45M ABA in the dark. Vertical bars represent standard errors (n=4).
This inhibition may result in, at least in part, the accu-mulation of ammonium in detached rice leaves dur-ing senescence in the dark. Another metabolic process producing ammonium is the reduction of nitrate. In our experiments, we observed that ammonium level was inversely correlated with nitrate level during senescence in darkness. If nitrate is assumed to be the
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Figure 7. Changes in nitrate level in detached rice leaves treated with
BA or ABA. Detached rice leaves were treated with either water, or 10M BA, or 45M ABA in the dark. Vertical bars represent standard errors (n=4).
source of ammonium, then detached rice leaves ought to accumulate more ammonium if fed with additional nitrate. In our work the exact result was observed. Detached rice leaves pretreated with 50 mM KNO3
for 24 h, followed by treatment with water for 24 h in the dark contained about 30% more ammonium than those pretreated with 50 mM KCl. Our results seem to suggest that ammonium accumulation during dark-induced senescence in attributed to a decrease in GS activity and an increase in nitrate reduction.
Our results indicate that ammonium accumulation is likely to participate in the regulation of senescence of detached rice leaves in the dark. This conclusion is based on the observations that (a) ammonium
accu-Figure 8. Changes in MDA level in detached rice leaves during
senescence in darkness and influence of NH4Cl on MDA level in detached rice leaves. For experiments of NH4Cl, MDA level was measured 3 days after treatment in darkness. Vertical bars represent standard errors (n=4).
Figure 9. Influence of STS on NH4Cl- and MSO-promoted senes-cence of detached rice leaves. The concentrations of NH4Cl, MSO and STS were 100, 0.5 and 0.2 mM, respectively. Protein level was determined 3 days after treatment in the dark. Vertical bars re present standard errors (n=4).
mulation paralleled the senescence under dark condi-tion; (b) detached rice leaves fed with NH4Cl or MSO,
which resulted in an accumulation of ammonium in leaves, promoted senescence; (c) treatments such as light and BA, which retarded senescence, decreased ammonium level, and (d) ABA, which promoted senes-cence, increased ammonium level.
Inhibition of GS activity in dark-induced, MSO-and ABA-treated detached rice leaves may also result in a decrease in glutamine level. Hurst et al. [8] report-ed that glutamine depletion rather than ammonium accumulation could be the reason for the reduced shelf-life of asparagus treated with phosphinothricin, an
inhibitor of GS. In our work, addition of glutamine had no effect on dark-induced, MSO- and ABA-promoted senescence (data not shown), it seems unlikely that lack of glutamine is the reason for the senescence of detached rice leaves in the dark or treated with MSO and ABA.
In the present study, we show that STS inhibits NH4Cl- and MSO-promoted senescence in detached
rice leaves. It seems that ammonium accumulated in detached rice leaves increases tissue sensitivity to ethylene, which in turn results in senescence promo-tion.
Acknowledgement
This work was supported financially by the National Science Council of the Republic of China (NSC 85-2321-B002-091) to C. H. K. Part XXXVI of the series “senescence of rice leaves”.
References
1. Berger MG and Fock HP (1983) Effect of methionine sulfox-imine and glycine on nitrogen metabolism of maize leaves in the light. Aust J Plant Physiol 10: 187–194
2. Beyer SM Jr (1976) A potent inhibitor of ethylene action in plants. Plant Physiol 58: 268–271
3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 4. Feller U and Fischer A (1994) Nitrogen metabolism in
senesc-ing leaves. Crit Rev Plant Sci 13: 241–273
5. Givan CV (1979) Metabolic detoxification of ammonia in tis-sues of higher plants. Phytochemistry 18: 375–382
6. Heath RL and Packer L (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid perox-idation. Arch Biochem Biophys 125: 189–198
7. Hecht U and Mohr H (1990) Factors controlling nitrate and ammonium accumulation in mustard (Sinapis alba) seedlings. Physiol Plant 78: 379–387
8. Hurst PL, King GA and Borst WM (1993) Postharvest inhi-bition of glutamine synthetase activity with phosphinothricin reduces the shelf-life of asparagus. Posthavest Biol Technol 3: 327–334
9. Kao CH (1978) Senescence of rice leaves II. Antisenescent actions of cytokinins. Proc Natl Sci Council ROC 2: 391–398 10. Kao CH (1980) Senescence of rice leaves IV. Influence of benzyladenine on chlorophyll degradation. Plant Cell Physiol 21: 1255–1262
11. Kao CH and Yang SF (1983) Role of ethylene in the senescence of detached rice leaves. Plant Physiol 73: 881–885
12. Kar M and Feierabend J (1984) Changes in the activities of enzymes involved in amino acid metabolism during the senes-cence of detached wheat leaves. Physiol Plant 62: 39–44 13. Lauriere C and Daussant J (1983) Identification of ammonium
dependent isoenzyme of glutamate dehydrogenase as the form induced by senescence or darkness stress in the first leaf of wheat. Physiol Plant 58: 89–92
14. Lea DJ and Miflin BJ (1974) Alternative route for nitrogen assimilation in higher plants. Nature 251: 614–616
15. Lin CC and Kao CH (1996) Disturbed ammonium assimilation is associated with growth inhibition of roots in rice seedlings caused by NaCl. Plant Growth Regul 28: 199–329
16. Liu J, Mukherjee I and Reid DM (1990) Adventitious rooting in hypocotyls of sunflower (Helienthus anus L.) seedlings. III. The role of ethylene. Physiol Plant 78: 268–276
17. Oak A, Stulen I, Jones K, Winspear MJ and Boesel IL (1980) Enzymes of nitrogen assimilation in maize roots. Planta 148: 477–484
18. Peeters KMU and Van Laere AJ (1992) Ammonium and amino acid metabolism in excised leaves of wheat (Triticum aestivum) senescing in the dark. Physiol Plant 84: 243–249
19. Platt SG and Anthon GE (1981) Ammonium accumulation and inhibition of photosynthesis in methionine sulfoximine treated spinach. Plant Physiol 67: 509–513
20. Platt SG and Rand L (1982) Methionine sulfoximine effect on C4plant leaf discs: comparison with C3species. Plant Cell Physiol 23: 917–921
21. Postius C and Jacobi G (1976) Dark starvation and plant metabolism. VI. Biosynthesis of glutamic acid dehydrogenase in detached leaves of Cucurbita maxima. Z Pflanzenphysiol 78: 133–140
22. Ronzio RA, Rowe WB and Meister A (1969) Studies in the mechanism of inhibition of glutamine synthetase by methion-ine sulfoximmethion-ine. Biochemistry 8: 1061–1075
23. Rowe WB and Meister A (1970) Identification of L-methionine-S-sulfoximine as the convulsivant isomer of methionine sulfoximine. Proc Natl Acad Sci USA 66: 500– 506
24. Simpson RJ and Dalling MJ (1981) Nitrogen redistribution dur-ing grain growth in wheat (Triticum aestivum L.). III. Enzymol-ogy and transport of amino acids from senescing flag leaves. Planta 151: 447–456
25. Storey R and Beevers L (1978) Enzymology of glutamine metabolism related to senescence and seed development in the pea (Pisum sativum L.). Plant Physiol 61: 494–500
26. Streit L and Feller U (1983) Changing activities and different resistance to proteolytic activity of two forms of glutamine synthetase in wheat during senescence. Physiol Veg 21: 103– 108
27. Strother S (1988) The role of free radical in leaf senescence. Gerontology 34: 151–156
28. Thimann KV (1980) The senescence of leaves. In: Thimann KV (ed) Senescence in Plants, pp 85–115. Boca Raton: CRC Press Inc
29. Thomas H (1978) Enzymes of nitrogen mobilization in detached leaves of Lolium temulentum during senescence. Planta 142: 161–169
