Accumulation and stability of nitrite in intact aerial leaves

Plant Science Letters, 11 (1978) 285--291

285

© Elsevier/North-HollandScientific Publishers Ltd.

ACCUMULATION AND STABILITY OF NITRITE IN INTACT AERIAL LEAVES

R I C H A R D W Y N J O N E S and R O B E R T W. S H E A R D

Department of Land Resource Science, University of Guelph, Guelph, Ontario (Canada, N I G 2W1)

(Received September 23rd, 1977} (Revision received and accepted November 29th, 1977)

SUMMARY

Nitrite accumulates in situ in nitrate reductase-containing green leaves switched from an aerobic to an anaerobic atmosphere in darkness, indicating that light is not necessary to NO~ reduction. Rates of accumulation in situ are only 10 to 20% of rates of exogenous NO~ accumulation by leaf slices in aqueous media. Furthermore, in wheat in situ accumulation is completed early compared to the aqueous system. These discrepancies may arise from the release of inhibitor(s) of NO~ reduction in the aqueous system. Nitrite does not accumulate in situ in the light, and dark anaerobically accumulated NO~ declines rapidly under illumination in either air or helium. These features are expected to arise from the coupling of NO~ reduction to primary photosynthetic electron transport. Dark aerobic decline of anaerobically accumulated NO~ occurred under the given conditions with a first order rate constant that was 7.6% of light~lependent decline, providing evidence under authentic conditions of NO~ metabolism in leaves in the absence of light.

INTRODUCTION Green leaves which contain nitrate reductase appear to display little if any NO~ assimilation in dark aerobic conditions [1--3]. Light is necessary for rapid NO~ reduction [4,5]. Its role has been conceived as indirectly supplying photosynthate and reductant via glycolysis to cytoplasmic nitrate reductase [6]. Alternatively, a direct and obligatory coupling of light to nitrate reduction may exist [3,7]. Contrary to uncertainty about the role of light in NO~ reduction, there is general agreement that NO~ reduction is closely coupled to photosynthetic electron transport [4,5,8], but there is uncertainty about the extent of exogenous NO~ reduction in aerobic leaf segments in darkness [3,6,7,9]. Leaf

286 segments also reduce NO] and accumulate exogenous NO~ during dark anaerobic incubation in aqueous media [ 6 , 7 , 1 0 , 1 1 ] , b u t this evidence for an indirect coupling o f light to nitrate reduction [6] is dismissable on the grounds of not being representative o f the intact leaf [ 3,7 ]. Furthermore, Canvin and Atkins [3] failed to detect in situ NO~ accumulation in intact leaf sections made aerially anaerobic. The apparent contrast b e t w e e n the behaviour o f leaf segments in aqueous media and o f relatively intact leaves in anaerobic gaseous environments w o u l d seem to support the hypothesis [3,7] that light is someh o w directly coupled to NO] reduction. We here present evidence that NO~ reduction does in fact occur in darkness in intact, aerial leaves when t h e y are made anaerobic, indicating that factors other than the absence o f light cause a suspension o f NO] reduction in the dark aerobic leaf. Anaerobically accumulated NO~ declines after transferring leaves to air in the dark. This decline constitutes evidence for NO~ metabolism under relatively authentic conditions of intact leaves containing NO~ generated in situ. MATERIALS AND METHODS

Wheat (Triticum aestivurn L. cv. Frederick), field peas (Pisum arvense L. cv. Century), maize (Zea mays L. cv. U.H. 108), and marrow (Cucurbita pepo L. cv. Cocozelle) were grown in vermiculite containing 1/10 Hunter's solution with 5 mM Ca(NO3)2 as described [ 1 2 ] . Growth temperature was 25 ° C and c o n t i n u o u s light o f 40 klux was provided b y Sylvania Gro-Lux fluorescent tubes. Plants were placed in darkness 30 rain prior to an experiment. Thereafter manipulations except for light treatment were performed under dim green safelight providing ~ 0 . 5 ~E • m -2 • sec -1. First leaves of 7
287 TABLE I DARK ANAEROBIC NITRITE ACCUMULATION IN LEAVES Species

Conditions

NO~ (nmol • g fresh wt -1 )

Peas

D, D, D, D,

0.5 59.2 7.0 52.1

Marrow

air He air He

± ± ± ±

0.1 2.9 3.6 12.5

Maize

D, air D, He

7.1 + 0.7 15.5 + 1.4

Wheat

D, air

4.1 90.5 5.2 4.3

D, He L, air L, He

+ + + ±

0.5 6.1 0.5 0.6

Means are followed by their standard errors. Immediately after excision, the leaves received 5 min air or He in the dark (D) or, for wheat, in light (L), as detailed in the text.

,

I

I

'

~.~ 240

tt3"2;.

11

g 160

-tl .6 -~

X ~"

0 r, 0

,

10

I

20 Time (min)

'

i

30

i

0

40

Fig. 1. Time course of in situ anaerobic accumulation and aerobic decline (solid lines) of NO~ in intact wheat leaves. Accumulation and decline were followed in separate experiments, the latter after prior anaerobic incubation for 10 and 20 rain followed by transfer of leaves to an open beaker (other details as in t e x t ) . . , anerobic incubation; v, aerobic incubation. The rates of in situ accumulation are compared with rates of exogenous NO~ accumulation (broken lines) by transverse leaf slices (1 m m deep, 50 mg wt) incubated in 5 ml 50 mM phosphate buffer (pH 7.5) at 25 ° C. Air was displaced from the samples by bubbling He through for 2 min, followed by quick stoppering of the text tubes.

288

(Table I) or in leaf analyzed straight from the plant. Large increases in NO~ ranging from 7- to > 100-fold occurred in pea, marrow, and wheat leaves made anaerobic; maize was exceptional in providing only a 2-fold increase (Table I), and no increase was detected in the primary leaf. The slight lag indicated for NO~ accumulation in wheat leaves (Fig. 1) may result from incomplete anaerobiosis during the first 5 min of He treatment. Nevertheless, rates of NO~ accululation were only 10 to 20% of those encountered with these species in vacuum-infiltrated [11] and He-bubbled (Fig. 1) aqueous media devoid of exogenous NO]. Moreover, in wheat in situ accumulation reached a plateau by 30 min in contrast to a continuing NO~ increase in the aqueous system. The accumulation of NO~ depicted in Table I and Fig. 1 is thought to be a consequence of NO~ reduction, since it neither occurs in leaves of plants grown without nitrate (lacking both NO~ and nitrate reductase), nor in plants receiving NO~ with tungsten (containing NO] but no active nitrate reductase [8] ). As expected from tight coupling to photosynthetic electron transport, light entirely prevented NO~ accumulation in anaerobic wheat leaves (Table I). Accumulated NO~ in aerial leaves provided the means to test for dark aerobic NO~ metabolism under authentic conditions. This test does not suffer from the ambiguity of assuming that exogenous NO~, applied to leaf segments, reproduces the behaviour of NOT generated intracellularly [3,6,7,9]. Wheat leaves containing NO~ were made aerobic, and the decline on NO~ (Fig. 1) shows that I

I

I

I

I

4.5

8 3.5

8 ,z

2.5

,3 I

0

0.5

I

1.0

|

I

I

I

I

I

1.5

2.0

0

0.5

100

1.5

2.0

Time (min) Fig. 2. First order decay kinetics o f NO~ in light-exposed wheat leaves in He (A) and air (B). Leaves were incubated anaerobically in darkness for 10 and 20 rain. Light was then turned on in (A); a further 2 min o f air flow was allowed in (B) before light was turned on. Timing starts with light on. Further details as in text. The rate constants (min-z ) in (A) are 1.00 (high NO~) and 0.97 (low NO~), and in (B) 0.90 (high NO~) and 0.99 (low NO~), giving a mean and standard deviation of 0.97 ± 0.05.

289 NO~ metabolism did in fact occur. Nitrite declined in an apparent first order reaction with a rate constant of 0.074 + 0.002 min -1 for two initial NO~ levels. Nitrite was very labile in the light {Fig. 2), yielding first order decay with average rate constant of 0.97 + 0.05 min-' for two initial NO~ levels and gaseous environments. DISCUSSION The foregoing experiments show that NO] reduction can occur in darkness w h e n intact aerial leaves are made anaerobic. Therefore the absence of NO~ reduction in dark aerobic leaves is not necessarily a consequence of the absence o f light. That it is also not merely a consequence of thiol-reversible ADP inhibition, where thiols increase in the light and ADP in the nitrate reductase compartment declines [13], is suggested by expected increases in cytoplasmic ADP concentration in dark anaerobic conditions. Klepper et al. [6] have provided evidence that the source of electron d o n o r for NO~ reduction is glyceraldehyde3-phosphate oxidized during glycolysis. The dark anaerobic leaf and the light aerobic leaf m a y be proposed to have in c o m m o n an increased supply o f this glycolytic intermediate which can in turn facilitate NO~ reduction. In the f o r m e r case, this increase can arise from removal of the Pasteur inhibition of glycolysis by anaerobiosis [14]. The NADH produced by glycolytic oxidation can be further increased by elimination o f respiratory competition [14,15]. In the light, photosynthesis can directly contribute glyceraldehyde-3-phosphate [6,16], either where net CO~ fixation is involved or where oxidized glycolytic intermediates are reduced via cytoplasmic/chloroplastic shuttles. Furthermore, mitochondrial respiration m a y be suppressed in the light under aerobiosis [ 17]. In contrast to another report [3], we find that the behaviour o f excised leaves in anaerobic gaseous environments is not qualitatively different from that of leaf discs and slices in anaerobic aqueous environments. Nitrate reduction in the latter circumstance would not seem to issue from the artificiality o f segmented leaves in aqueous media. The quantitative differences between the two systems is important, however, and could arise by release of inhibitor(s) from segments into solution, where these are not otherwise afforded exit from intact leaves in a gas. Nitrite inhibits the assimilation o f NO~ in leaves [3] and causes an apparently irreversible inhibition o f Chlorella nitrate reductase in vivo [ 18]. It specifically b u t weakly inhibits spinach nitrate reductase in vitro [ 1 9 ] . The high Ki (7.5 mM) o f in vitro inhibition might seem to make this reaction irrelevant, since aerial wheat leaves attain a NO~ concentration o f only ca 0.4 mM after prolonged anaerobic incubation (Fig. 1). However, this measured concentration is distributed over t h e whole leaf, whereas in reality it m a y be envisaged to be m u c h higher in the vicinity of nitrate reductase, resulting from such concentrating effects as (a} presence of e n z y m e and p r o d u c t in the extra-vacuolar space, and (b) absence of e n z y m e and product from the vascular tissues and possibly also from chlorophyll-free epidermal cells. The decline u n d e r illumination o f c o m p o u n d s which inhibit NO~ reduc-

290

tion, such as NO~ [19] and also ADP [13,16,21], is a way by which light can stimulate NO~ assimilation in anaerobic aerial leaves [ 3,7 ] by means of a v e r y indirect coupling. Under the given experimental conditions, and for a given NO~ level, NO~ under dark aerobic conditions disappeared with a rate constant that was 7.6% o f that in the light. This value represents something o f a m a x i m u m estimate o f dark relative to light aerobic NO~ metabolism, since significant NO~ reduction occurs in the light but n o t in the dark [1,2,5]. Nitrite disappearance is frequently taken as a sign o f NO~ reduction [6,7,9], but in view o f possible oxidation back to NO~ in aerobic conditions [ 2 0 ] , this evidence for reduction is somewhat ambiguous. Canvin and Atkins [3] did show, with an open aqueous system, that NO~-N after an hour's delay was incorporated into reduced N with about the same rate relative to light as in situ NO~ disappearance recorded here. T h e y used vacuum infiltration to make the tissue accessible to NO~, and since this technique appears to make leaf segments initially quite anaerobic [ 11], it is likely that the lag-free aerobic decline in NO~ which we detect is in fact due to reduction. The proper inference to be made from similar kinetics o f NO~ disappearance in the light under aerobic or anaerobic conditions is as yet uncertain. Given that the rate o f NO~ reduction is many-fold higher than NO~ reduction, and that the latter in the short-term is insensitive to gaseous composition [3], NO~ reduction would seem to be independent o f CO2 fixation. However, there m a y be no conflict with a recent study indicating the regulation o f NO~ reduction by CO2-fixation products [22], since in the current study NO~ disappeared within a time period possibly insufficient for the build-up of these products. This study furnishes independent corroboration of a well-known observation, namely that NO~ does not ordinarily accumulate in leaves. This is probably because NO~ reduction represents the limiting step in NO~ assimilation [4,5] w h e t h e r leaves are in the dark or in the light. ACKNOWLEDGEMENT

This w o r k was financially supported b y the National Research Council of Canada. REFERENCES 1 2 3 4 5 6 7 8 9

H. Burstr~m, Ann. Agric. Coll. Sweden, 11 (1943) 1. V. Stoy, Physiol. Plant., 8 (1955) 963. D.T. Canvin and C.A. Atkins, Planta, 116 (1974) 207. L. Beevers and R.H. Hageman, Annu. Rev. Plant Physiol., 20 (1969) 495. L. Beevers and R.H. Hageman, in A.C. Giese, (Ed.), Photophysiology, Vol. 7 Academic Press, London, 1972 p. 85. L. Klepper, D. Flesher and R.H. Hageman, Plant Physiol., 48 (1971) 580. C.A. Atkins and D.T. Canvin, Planta, 123 (1975) 41. E.J. Hewitt, Annu. Rev. Plant Physiol., 26 (1975) 73. J.W. Radin, Plant Physiol., 51 (1973) 332.

291 10 11 12 13 14 15 16 17 18 19 20

E.G. Jaworski, Biochem. Biophys. Res. Commun., 43 (1971) 1274. R.W. Jones and R.W. Sheard, Can. J. Bot., 55 (1977) 896. R.W. Jones and R.W. Sheard, Can. J. Plant Sci., 53 (1973) 207. A.R.J. Eaglesham and E.J. Hewitt, Plant Cell Physiol., 15 (1975) 1137. R.W. Jones and R.W. Sheard, Plant Physiol. Suppl., 59 (1977) 72. A.J. Reed and R.H. Hageman, Plant Physiol. Suppl., 59 (1977) 127. U. Heber, Annu. Rev. Plant Physiol., 25 (1974) 393. B.S. Mangat, W.B. Levin and R.G.S. Bidwell, Can. J. Bot., 52 (1974) 673. E.K. Pistorius, H.-G. Gewitz, H. Voss and B. Vennesland, Planta, 128 (1976) 73. A.R.J. Eaglesham and E.J. Hewitt, Biochem. J., 122 (1971) 18 p. E.J. Hewitt and T.A. Smith, in Plant Mineral Nutrition, English Universities Press, 1975, pp. 219--222. 21 N. Nelson and I. ]]an, Plant Cell Physiol., 10 (1969) 143. 22 Z. Plaut, K. Lendzian and J.A. Bassham, Plant Physiol., 59 (1977) 184.