Planta (1982)156:226~232

P l a n t a 9 Springer-Verlag 1982

Utilization of nitrite and nitrate by dwarf bean Hans Breteler* and Wieslaw Luczak** Research Institute ITAL, P.O. Box 48, NL-6700 AA Wageningen, The Netherlands

Abstract. The uptake and conversion of N O f and the effect of N O f on the uptake and reduction of N 0 3 were examined in N-depleted Phaseolus vulgaris L. Nitrite uptake at 0.1 mmol d m - s was against an electrochemical gradient and became constant after one or two initial phases. Steadystate uptake declined with increasing ambient NO 2 concentration (0-0.7 mmol din-3). In this concentration range root oxygen consumption was unaffected by N 0 ~ , indicating that the decrease of NO~ uptake was not related to respiration. After 6 h N 0 2 supply, about one-third of the absorbed NO~- had accumulated, mainly in the root system. Oxidation of N 0 2 to NO 3 was not observed. The apparent induction period for NO 3 uptake was about 6 h in control plants and 3.5 h in plants that were pretreated for 18 h with NO~. In contrast, the time course of NO 2 uptake was unaffected by pretreatment with N 0 3. Steadystate NO 3 uptake was less affected by NO 2 than was steady-state NO 2 uptake by NO 3. Nitrate reductase activity (NRA) in leaves and roots was induced by both NO~ and NO~. In roots, induc9 tion with NO 2 was faster than with NO]-, but there was no difference in N R A after 5 h. Nitrite inhibited N R A in the roots of N 0 3-induced plants and thus seems to stimulate the induction, but not the activity of induced nitrate reductase. In view of the observed differences in time course and mutual competition, a common uptake mechanism for NO 2 and NO 3 seems unlikely. Expression of the NO 2 effect on the induction of NO 3 uptake re-

* To whom correspondence should be addressed Botanical Garden, Polish Academy of Sciences, ul. Prawdziwka 2, PL-02-927 Warsaw, Poland

** Permanent address: Abbreviations:

NR(A) = nitrate reductase (activity); BM = basal

medium

0032-0935/82/0156/0226/$01.40

quired more time than the induction itself. We therefore conclude that NO~ is not the physiological inducer of NO 3 uptake. Key words: Nitrate (uptake) - Nitrate reductase - Nitrite uptake - Phaseolus (NO~- and NO 2 uptake). Introduction

Nitrite is an obligatory intermediate in the assimilation of NO 3 and is usually present at low concentrations in NO3-grown plants (Beevers and Hageman 1969). An oxidative pathway of NO~ synthesis also occurs in biological tissues (Tannenbaum et al. 1978), but claims for the oxidative formation of NO 2 and NO 3 in higher plants are generally considered as insufficiently supported (Hewitt and Smith 1974). Exogenous NO~- may originate from the transformation of nitrogen compounds in the soil and rhizosphere (Van Veen and Frissel 1981 ; Smit and Woldendorp 1981), from organic wastes (Germon et al. 1979), or from NO~-containing roots during oxygen stress (Lee 1980). At high concentrations, NO 2- has been reported to be toxic for plants, especially at low pH (Hewitt 1966; Lee 1979). At lower concentrations, NO~- can serve as an Nsource and interfere with certain aspects of NO~utilization (Jackson 1978). In the framework of our investigation into NO 3 utilization by the dwarf bean, we have studied the utilization of NO~- and the effect of exogenous NO~ on two key steps in NO 3 utilization, i.e., NO 3 uptake and the reduction of NO 3 to NO~. Previous work has indicated the root system as the major site for these processes during the initial hours of NO~- utilization (Breteler and H/inisch ten Cate 1980). The present data allow

227

H. Breteler and W. Luczak: Utilization of nitrite and nitrate

a comparison, under identical conditions, between the utilization of nitrite and nitrate and their interaction at the onset of nitrogen nutrition.

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Materials and methods

Phaseolusvulgaris L. cv. Witte

Krombek was grown unnodulated in a nitrogen-free liquid basal medium at pH 5.0_+0.1. Plant cultivation and experiments were done at 20 ~ C, 65% relative humidity, and a 16-h photoperiod at 30 Wm 2: Detailed information about the growth conditions is given by Breteler et al. (1979). Experiments were started after 7 d by the addition of KNO2 or KNO3 to the N-free basal medium. UnIess stated otherwise, NO 2 was given at 0.1 and NO3, at 0.1 or 0.4 mmol dm -3, The difference between the latter concentrations did not affect the rate of NO3, utilization (Breteler and Nissen 1982). In one experiment, I mmol dm -3 o f a pH 7.5 phosphate buffer was added to basal medium in order to study NO z uptake at that pH. Uptake of NO 3- and NO~ was measured by ambient depletion. Nitrate reductase activity was measured by an anaerobic in vivo procedure (Breteler et al. 1979), which yielded results concurring with the rate of actual reduction (Breteler and Hfinisch ten Cate 1980). The value of the nitrate reductase activity in NO~--free tissues after NO~ pretreatment was corrected for NO 2 release during incubation by subtraction of the activity in sulfate buffer from that in nitrate buffer (Breteler et al. 1979). Nitrate was determined colorimetrically in media and in plant extracts with Szechrome reagent (R & D Authority Ben Gurion University of the Negev, Israel) or as NO 2 after hydrazine reduction (Breteler and H/inisch ten Cate 1980). Nitrite was assayed in extracts and in media after diazotation, as described for the nitrate reductase activity test. Extracts were prepared from fresh tissues powdered in liquid N 2 and homogenized for 30 s with sodium dodecylsulfate (1% w/v) in a Virtis 45 homogenizer. The crude extract was centrifuged (10 min, 2 ~ C, 12,000 g) and the supernatant of root extracts was assayed directly. The supernatant of shoot extracts was extracted 3 x with trichloroethylene to remove green pigments, and the aqueous phase was centrifuged again. Oxygen uptake by the roots was determined polarographically (H/inisch ten Cate and Breteler 1981). The electrical potential difference between vacuoles of root cortical cells and the medium was measured as described by Macklon and Higinbotham (1968). Treatments comprised of two replicates with at least three plants each. Experiments were repeated at least twice and data from representative experiments are given. Unless stated otherwise, the results are expressed per unit of dry root mass.

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I

Results

Nitrite uptake. After a rapid start, initial NO 2 uptake slowed down and became constant after 2-5 h (Fig. 1). At low concentrations the rapid phase consisted of one or two bumps. Steady-state uptake declined progressively with the NO 2 concentration and became zero above 700 tamol din-3 (Fig. 2). Under alkaline conditions (pH 7.5), net NO2 uptake ( 1 0 0 g m o l d m -3) occurred for at least 6 h. Nitrate, either supplied concomittantly with NO~ (Fig. 3) or as a pretreatment (7 18 h at 2 mmol din-3, data not shown), did not affect

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228

H. Breteler and W. Luczak: Utilization of nitrite and nitrate

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Fig. 4. Effect of NO 3 on NO2 uptake by dwarf bean. Nitriteinduced (4 h, 0.1 mmol dm-3) plants were supplied with NO~(0.4 mmol dm-3) for 1 h as indicated by a r r o w s . Nitrate was removed from the roots by two washes (1 min each) with fresh NO2- medium (o) and the NO~ concentration between 2 and 3 h was below 1 Ixmoldm-3. In control plants (o) the medium was not changed after 2 h

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Fig. 5. Rate of NO 3 uptake by dwarf bean. Plants were pretreated with NO 2 (18 h, 0.1 mmol dm -3, (o)) or kept on basal medium (e) before the start of NO~ nutrition (0.4 mmol din-3). Absolute rates after 6 h:26.1 + 2.5 (control) and 32.1 _+3.3 pmol h - ~ g- t (NO 2 pretreatment)

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1. Nitrite content and nitrite balance in dwarf bean during the initial 6 h of nitrite supply (0.1 mmol din-3). Contents are given per g dry matter of shoot and roots, respectively. Balance data per g dry root and (in parenthesis) as % of uptake

Table

Time

Contents

Balance

Shoot

Uptake

Roots

2:~0

-4--

Accumulation

(pmol g- 1)

(pmol g- 1 dry matter of roots)

0 2 4 6

0 2.0 2.5 3.3

48.6

16.5 (34)

20 2-

Reduction

(h)

0 8.6 10.6 10.6

3

32.1 (66)

the time course of NO2 uptake, but after 5 h of concomittant supply, NO~ uptake was only 10% lower in the presence of NO~ (Fig. 3). Nitrite was lost from NO~-induced plants upon the provision of NO~- (Fig. 4). However, NO 2 uptake recovered after 80 rain and was enhanced after removal of NO2. Within 7 h after of the addition of NO 2 root respiration was not significantly altered at concentrations up to 1 mmol dm -3. Only at an extreme concentration of nitrite (25 mmol dm-3), was there a marked decrease in the consumption of oxygen by the roots (data not given). Fate of nitrite. Plants kept for one week on basal medium were flee of NO~ and NO~, the limit of detection being 60 and 100 nmol per g dry tissue,

0

1

2

3

Fig. 6. Effect of NO~ on NO~ uptake by dwarf bean. Nitrateinduced plants(18 h, 0.4 mmol dm -a) were supplied with NO~ (0.1 mmol dm -3) for i h as indicated by a r r o w s . Nitrite was removed From the roots by two washes (1 min each) with fresh NO 3 medium. The NO= concentration between 2 and 3 h was below 2 pmol dm 3

respectively. After 2 h of N O ; supply the mean concentration of NO~ in tops and roots approximated 0.2 and 0.5 mmol dm -3, respectively, thus exceeding the ambient NO2 level of 0.1 mmol d m - 3 (Table 1). After 6 h most of the plant's N O ; was present in the roots and NO 2 accumulation represented about one third of total NO2 absorption (Table 1). Two-thirds of the absorbed NO2 were not recovered, as such, after 6 h which corresponds to an average rate of N O ; reduction in whole plants of about 5.4 pmol NO 2 h - a g- 1 root. Fresh extracts of roots and tops from NO2pretreated plants (2-18 h, 0.1 mmol dm-3) did not contain any NO~-, and the NO~ content during the initial 24 h of NO~- supply (2 mmol dm -3) never exceeded 0.5 and 0.4 pmol per g dry root and top, respectively (data not shown).

H. Breteler and W. Luczak: Utilization of nitrite and nitrate

229

Table 2. Nitrate reductase activity (NRA) in primary leaves and roots of dwarf bean after 18 h pretreatment with nitrite (0.1 mmol din-3). NRA was assayed either in N O ; buffer or in SO]- buffer. Data are expressed as nmol NO 2 h -1 leaf disk -~ or gmol NO~- h ~ g-a dry root, and given • (N=5). BM=basal medium Pretreatment

BM(control) BM +NO2

Leaves

Roots

NO;

SOl-

NO3-SO 2

NO~

SOl-

NO 3-SO ] -

17.5• 33.9•

1.4+1.5 3.2•

16.1• 30.7•

0.20• 3.18•

0.16• 1.15•

0.04• 2.03•

Table 3. Nitrate reductase activity (NRA) in roots of dwarf bean in the course of nitrate or nitrite nutrition. N-sources 0.1 mmol dm 3. NRA was assayed either in NO~- buffer or in SOl - buffer. Data are given • SD (N= 4) and expressed as pmol NO~- h - z g- i

N-source

NO; NO;

1h

5h

NO;

SOl-

NO;-SO]-

NO;

SOl

NO;-SO~-

0.8• 4.9•

0.7• 2.0•

0.1• 2.9•

6.1• 6.6•

6.4• 5.1•

-0.3• 1.5•

Effect of nitrite on nitrate uptake. There was an apparent induction period of about 6 h for NO2 uptake by N-depleted plants (Figs. 3, 5). This period was shortened by several hours after 18 h pretreatment with NO2 (Fig. 5). Pretreatments shorter than 12 h were ineffective in this respect (data not shown). After NO2 pretreatment NO~- was taken up at a 10 to 20% higher rate than after pretreatment with BM (cf. Subscript Fig. 5). A concomittant supply of NO~- and NO 3 inhibited NO~- uptake by about 40%, leaving the time course of uptake unaffected (Fig. 3). Addition of NO 2 to NO3-induced plants inhibited NO 2 uptake, but the inhibition was almost completely overcome after 20 min (Fig. 6). Removal of NO2 after i h, however, resulted in 60% lower NO 2 uptake than before the addition of NO~-. Uptake remained constant when NO 2 was left in the medium (data not shown). Effect of nitrite on nitrate reductase activity. Substantial N R A was induced in leaves and roots after 18 h pretreatment with NO 2 (Table 2). The difference between the root N R A of NO~-pretreated and control plants vanished after 2 to 4 h of NO 2 supply (data not shown). Nitrate-free dwarf bean leaves, in contrast to roots, had much more potential (NO2 buffer) than actual (SOl- buffer) N R A (Breteler et al. 1979). After 1 h nitrogen supply, NO~- induced a higher N R A in the roots than did NO 3- (Table 3). This difference disappeared during the subsequent hours. In NO 2-induced plants root NRA was inhibited after the addition of NOs (Table 4). Control plants had constant N R A during

Table 4. Nitrate reductase activity (NRA) in roots of dwarf bean in the course of nitrite supply (0.1 mmol dm 3) to NO~induced (18 h, 2 mmol dm 3) plants. NRA was assayed either in N O ; buffer or in SO4z- buffer. Data are given • SD (N= 5) and expressed as gmol NO~ h - 1 g 1 Time (h)

NO;

SOl-

0 2 4

18.4• 15.9• 13.6•

17.7• 14.9• 13.7•

NO;-SO]0.7• 1.0• -0.1•

the experimental period (cf. H/inisch ten Cate and Breteler 1981). Discussion

Nitrite uptake. The time course for NO~- uptake differs from that of other ions, including NO 2. At low concentrations (< 100 tamol dm -3) uptake increased temporarily (once or twice), while a decrease was followed by a constant rate at higher concentrations (Fig. 1). This unusual uptake pattern was also observed in wheat (Jackson et al. 1974a) and indicates that NO2 may be absorbed by an unknown NO~- -specific mechanism. At pH 5 about 2% of the added NO 2 is in the form of nitrous acid, the rest being nitrite anion (pKa HNO2 = 3.37). The speciation at the common pH of cell sap (Thomas et al. 1973) is even closer to NO~-. Nitrite toxicity is believed to be due to undissociated H N O 2 rather than to NO 2 (Lee 1979). Accordingly, this would imply that NO~- uptake ceases at 15, and that root respiration is not hampered up to 20, but considerably curtailed at

230 600 gmol HNO2 din-3. Nitrite was also taken up at pH 7.5, where the contribution of H N O 2 is negligible. We therefore conclude that N O ; - N was mainly absorbed against the gradient of the NO 2 concentration (Table 1) as well as against an electropotential difference of - 4 0 mV between medium and root cells (data not shown). The cessation of NO 2 uptake beyond 700 txmol dm -3 (Fig. 2) was not caused by an inhibition of respiratory metabolism. Nitrite may affect other aspects of metabolism or could specifically restrict its own uptake. Plants with a fully-induced NO 3 uptake system had the same pattern of N O ; uptake as non-induced plants, indicating that initial NO 2 uptake did not benefit from the apparent induction of NO 3 uptake. Jackson et al. (1974b), on the other hand, observed induced NO 2 uptake after 12 h of NO~ supply to wheat. Steady-state NO 2 uptake was about half that of steady-state NO;- uptake when the N-sources were provided separately at equimolar concentrations (Fig. 3). Nitrite uptake was hardly inhibited by NO;- when present concomittantly at the start of N supply (Fig. 3). However, net NO 2 uptake became negative when NO~ was given to N O ; induced plants (Fig. 4). In the latter case the inhibition decreased with time and no traces of inhibition were left after removal of NO~-. The inhibition of N O ; uptake by NO2 thus seems to be reversible. Nitrite balance. Nitrite is absent from N-depleted plants and very low during NO;- nutrition. On a molar basis, accumulation of NO 2 during NO 2 nutrition is less than one-third of the accumulation of NO2 during NO2 nutrition under comparable conditions (Breteler and Hfinisch ten Cate 1980). In 6-h experiments about 33 and 75% of all the absorbed nitrogen accumulated as NO 2 or NO~ during NO 2 or NO~ nutrition, respectively. The mean rate of N O ; reduction in whole plants of 5.4 (32.1:6, Table 1) exceeded the rate of NO~ reduction of 3.3 gmol h -a g - t root found by Breteler and H/inisch ten Cate (1980) under NO~ nutrition. The difference between the accumulation of NO~ and N O ; indicates that NO 2 reduction also exceeded NO~ reduction during NO;- nutrition. The absence of detectable NO~ in NOz-pretreated plants indicates the absence of heterotrophic nitrification in our experiments. Synthesis of NO~ has been reported in Ankistrodesmus braunii (Kessler and Oesterheld 1970; Spiller et al. 1976) as well as in higher plants (Freny6 and Mihfi-

H. Bretelerand W. Luczak: Utilizationof nitrite and nitrate lyfi 1970; Muhammad and Kumazawa 1974; Sahulka and Lisfi 1978; Funkhouser et al. 1981). The data for higher plants, however, are generally hard to reproduce. The oxidative pathway may, at least in part, operate post mortem (Freny6 1966). Other authors found no evidence for an oxidative formation of N O ; or NO3 in algae (Ohmori 1978 and references therein). A strict proof of NO;- formation would require well-conserved extracts of sterile, fresh plant tissue, the reproducible demonstration of sizeable amounts of 15NO~- from a reduced 15N precursor, and a characterization of the enzyme or chemical mechanism involved. Inhibitors of the assimilatory higher plant NR could be a useful tool in these efforts, provided they do not interfere with the possible oxidation process itself. The absence of incontrovertible evidence for heterotrophic nitrification indicates that the phenomenon, if it exists, is not ubiquitous in the nitrogen metabolism of higher plants. Nitrate uptake. The apparent induction of NO 3 uptake was completed about 2.5 h earlier in N O ; pretreated plants than in control plants (Fig. 5). The effect of N O ; on the time course of NO~uptake was time-dependent. At least 12 h of NO 2 supply were required to shorten the induction period, and the uptake pattern was not changed when the two N-sources were given simultaneously (Fig. 3). Moreover, the NO 2 content in roots was appreciable after 2 h of N O ; supply (Table 1), so that any effect of endogenous NO 2 could have been exerted from that time. The effect of NO 2 is not due to a changed N status of the plants because ammonium and the prevailing amino compounds of dwarf bean were unable to alter the time course of NO;- uptake (Breteler, in preparation). For these reasons it seems that a consequence of NO 2 metabolism rather than NO 2 itself accelerates the induction of NO2 uptake. It is likely that this "consequence" also occurs in the course of NO;- nutrition. An NOz-accelerated induction of NO 3 uptake in intact higher plants has not been reported before. Jackson et al. (1974b) and Tompkins et al. (1978) reported that NO 2 had no effect or delayed the induction of NO;- uptake in the roots. In cultured tobacco cells (Heimer 1975) and in a fungus (Schloemer and Garrett 1974), however, NO;- uptake was induced more rapidly after the provision of NO 2. Uptake of NO;- was reduced by about 40% when nitrogen was initially supplied as a NO 3 ~N O ; mixture (Fig. 3). Provision of N O ; to NO;-induced plants caused a short transient inhibition

H. Breteler and W. Luczak: Utilization of nitrite and nitrate

(Fig. 6) of NO 3 uptake. After 20 rain the uptake rate was only 15% lower. Remarkably, the inhibition of NO~- uptake was far more pronounced when NO 2 was removed from the root medium. The latter finding shows that endogenous NO 2 or its metabolism, rather than exogenous NO~-, inhibited NO~- uptake. It is unclear why this inhibition was more effective in the absence than in the presence of ambient N O ] . The observed pattern of inhibition is not due to an experimental artifact, because control plants were not affected in this respect by a short wash with fresh nitrate medium (see subscript Fig. 6). We recall that NO~uptake was reversibly inhibited by NO~- (Fig. 4). A common uptake mechanism for NO 2 and NO~seems unlikely in view of the different uptake-time patterns and the lack of symmetry in the mutual effects on uptake induction and induced uptake. On the basis of similar experiments, Jackson et al. (1974b) came to the same conclusion for wheat.

Nitrate reductase. Nitrate reductase in leaves and roots could be induced by NO~- as well as by NO 2 (Tables 2, 3, Breteler et al. 1979). Induction of root N R A proceeded faster with NO 2 than with NO~-, but the levels of enzyme activity were comparable 5 h after the onset of nitrogen supply. Similarly, root NRA in NO2-pretreated plants was the same as in control plants after 2-4 h of subsequent NO2 supply. Induction of NR by NO 2 has been reported for roots (Sahulka and Lisfi 1978) and some other organs of higher plants (Shen 1972; Lips etal. 1973; Kaplan etal. 1978). The absence of NO2-inducible N R A in other species (Beevers et al. 1965; Joy 1969), however, lead Hageman and Reed (1980) to conclude that NO 2 is not an inducer of NR in all species. Whether NR is induced in situ by its substrate or its product still awaits the proper assessment of the existence and relevance of heterotrophic nitrification. In bean roots NO 2 clearly is a more potent inducer than NO 2 . The activity of pre-existing enzyme was inhibited after a few hours of NO 2 supply (Table 4). Correction of N R A values in NO2-pretreated plants for the release of endogenous NO 2 (see Materials and methods) is justified by the absence of endogenous NO~- as a potential source of NO 2 release. It requires, however, that NO 2 release is the same in NO~-- and SO~- buffer. On this assumption we conclude that NO 2 promotes the induction, but not the activity per se of NR. In some algae N R A is also inhibited by NO 2 (Syrett and Morris 1963 ; Thacker and Syrett 1972).

231

NO~ Uptake-reduction association. Pretreatment of nitrogen-starved plants with NO~- enhanced the induction of two successive key steps in NO~ utilization, i.e., NO~- uptake (Fig. 5) and the reduction of NO 3 to NO 2 (Tables 2, 3). At least 11h elapsed, however, between the stimulation of root N R A and the acceleration of uptake by NO 2 . This time lag argues against a coordinated induction of uptake and NR, as was suggested by Heimer (1975) and Butz and Jackson (1977). In NO~--induced plants root N R A was inhibited within 2 h of NO 2 supply (Table 4). The NO 3 uptake rate was hardly affected after several h of mixed NO 2 + NO 3 nutrition (cf. Fig. 6), indicating that uptake and reduction in induced plants were also differentially affected by NO 2 . We gratefully acknowledge the analytical help of J. Broertjes, the root potential measurements by Dr. A.E.S. Macklon (Aberdeen) and the critical review of the text by Professor P. Nissen (Bergen). W.L. wishes to thank the Dutch Ministry of Science and Education and the Polish Academy of Sciences for the opportunity to spend three months in Wageningen.

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232 the study of plant nutrition. 2nd edn. Commonwealth Agricultural Bureaux, Farnham Royal Hewitt, E.J., Smith, T.A. (1974) Plant mineral nutrition. The English Univ. Press, London Jackson, W.A. (1978) Nitrate acquisition and assimilation by higher plants: processes in the root system. In: Nitrogen in the environment, pp. 45-88, Nielsen, D.R., MacDonald, J.G., eds. Academic Press, New York London Jackson, W.A., Johnson, R.E., Volk, R.J. (1974a) Nitrite uptake by nitrogen-depleted wheat seedlings. Physiol. Plant. 32, 37-42 Jackson, W.A., Johnson, R.E., Volk, R.J. (1974b) Nitrite uptake patterns in wheat seedlings as influenced by nitrate and ammonium. Physiol. Plant. 32, 108-114 Joy, K.W. (1969) Nitrogen metabolism of Lemna minor. II. Enzymes of nitrate assimilation and some aspects of their regulation. Plant Physiol. 44, 849-853 Kaplan, D., Mayer, A.M., Lips, S.H. (1978) Nitrite activation of nitrate reductase in higher plants. Planta 138, 205-209 Kessler, E., Oesterheld, H. (1970) Nitrification and induction of nitrate reductase in nitrogen-deficient algae. Nature (London) 228, 28%288 Lee, R.B. (1979) The effect of nitrite on root growth of barley and maize. New Phytol. 83, 615-622 Lee, R.B. (1980) Sources of reductant for nitrate assimilation in non-photosynthetic tissue: a review. Plant Cell Environ. 3, 65-90 Lips, S.H., Kaplan, D., Roth-Bejerano, N. (1973) Studies on the induction of nitrate reductase by nitrite in bean-seed cotyledons. Eur. J. Biochem. 37, 589-592 Macklon, A.E.S., Higinbotham, N. (1968) Potassium and nitrate uptake and cell transmembrane electropotential in excised pea epicotyls. Plant Physiol. 43, 888-892 Muhammad, S., Kumazawa, K. (1974) Assimilation and transport of nitrogen in rice. I. 15N_labelled ammonium nitrogen. Plant Cell Physiol. 15, 747-758

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