Planta
Planta (1990)182:149-154
9 Springer-Verlag1990
Nitrate and nitrite uptake and reduction by intact sunflower plants E. Agiiera, P. de la Haba, A.G. Fontes, and J.M. Maldonado* Departamento de BiologiaVegetaly Ecologia, Divisi6n de FisiologiaVegetal, Facultadde Ciencias,Universidadde C6rdoba, E-14004 C6rdoba, Spain Received 3 February; accepted 6 April 1990
Abstract. Nitrogen-starved sunflower plants (Helianthus annuus L. cv. Peredovic) cannot absorb NO 3 or NO~ upon initial exposure to these anions. Ability of the plants to take up NO 3 and NO~ at high rates from the beginning was induced by a pretreatment with NO 3 . Nitrite also acted as inducer of the NO~-uptake system. The presence of cycloheximide during NOf-pretreatment prevented the subsequent uptake of NO 3 and NO2, indicating that both uptake systems are synthesized de novo when plants are exposed to NOr. Cycloheximide also suppressed nitrate-reductase (EC 1.6.6.1) and nitrite-reductase (EC 1.7.7.1) activities in the roots. The sulfhydryl-group reagent N-ethylmaleimide greatly inhibited the uptake of NO 3 and NO 2. Likewise, Nethylmaleimide promoted in vivo the inactivation of nitrate reductase without affecting nitrite-reductase activity. Rates of NO 3 and NO~- uptake as a function of external anion concentration exhibited saturation kinetics. The calculated Km values for NO 3 and N O f uptake were 45 and 23 ~tM, respectively. Rates of NO~ uptake were four to six times higher than NOs-reduction rates in roots. In contrast, NOz-uptake rates, found to be very similar to NOs-uptake rates, were much lower (about 30 times) than NOz-reduction rates. Removal of oxygen from the external solution drastically suppressed NO 3 and NO~- uptake without affecting their reduction. Uptake and reduction were also differentially affected by pH. The results demonstrate that uptake of NO 3 and NO 2 into sunflower plants is mediated by energy-dependent inducible-transport systems distinguishable from the respective enzymatic reducing systems. Key words: Helianthus- Nitrate (reduction, u p t a k e ) Nitrite (reduction, uptake)
Introduction The utilization of inorganic nitrogen, either NO 3 or NO~, by higher plants involves the following steps: up* To whom correspondenceshould be addressed Abbreviations: CHI=cycloheximide; NEM=N-ethylmaleimide; NiR=nitrite reductase; NR=nitrate reductase; pHMB=phydroxymercuribenzoate
take of NO 3 or NO~ into the plant, their reduction to NH +, and incorporation of NH + to carbon skeletons to produce amino acids. Reduction of NO 3 to NH~proceeds by the sequential action of the enzymes nitrate reductase (NR) and nitrite reductase (NiR) (Guerrero et al. 1981). Ammonium is then assimilated mostly through the glutamine synthetase/glutamate synthase cycle (Lea and Miflin 1979). In various plants, the presence of NO 3 in the medium induces the development of a NOs-uptake system (Tompkins et al. 1978; Deane-Drummond 1984; MacKown and McClure 1988). A constitutive low-capacity uptake system for NO 3 has been found to be additionally present in barley plants which have not been previously exposed to NO 3 (Behl et al. 1988). In the diatom Phaeodactylum trieornutum (Cresswell and Syrett 1981) and in the cyanobacterium Anabaena cycadeae (Bagchi et al. 1985), the NO~--uptake system appears to be NH~-repressible rather than NOs-inducible. Recently, a protein of about 48 kDa has been detected in the plasma membrane of the cyanobacterium Synechococcus R2 grown on NO3, but not in NH~-grown cells (Maduefio et al. 1988). Likewise, the presence of NO 3 in the medium has been shown to induce the synthesis of 30- and 40-kDa proteins in the plasma membrane (Dhugga et al. 1988) and tonoplast (McClure et al. 1987) in corn roots. From these observations it has been suggested that the NO3-inducible membrane proteins might have a role in the uptake of NO3. Unlike NO 3 uptake, NO~ uptake by higher plants has been scarcely studied. The occurrence of a NO zuptake system has been only demonstrated in wheat (Jackson et al. 1974; Goyal and Huffaker 1986), dwarf bean (Breteler and Luczak 1982) and barley (Ibarlucea et al. 1983) seedlings. In sunflower plants, NR is specifically induced by NO 3 while NiR synthesis is enhanced both by NO 3 and NO z (Agfiera et al. 1987a; de la Haba et al. 1988). Aslam and Huffaker (1989) have recently reported that in barley leaves NiR is induced by NO 3 directly, i.e. without being reduced to NO 2, and that absorbed NO 2 induces the enzyme activity indirectly after being oxidized to NO 3 within the leaf. The present study was undertaken to investigate the existence of NO 3- and NOz-uptake systems in intact sunflower plants. As found in other plants, NO 3- and NO2-uptake systems
150 were s u b s t r a t e - i n d u c i b l e . Both u p t a k e systems have been characterized, s h o w i n g different properties f r o m those exhibited b y the respective r e d u c i n g systems.
Material and methods Plant culture. Helianthus annuus L. cv. Peredovic (supplied by Eurosemillas, C6rdoba, Spain) was germinated and grown on a 1:1 (v/v) mixture of perlite and vermiculite in a growth chamber with 16 h of light daily (200 ~mol. m - 2. s- x of photosynthetically active radiation) and a day/night regime of 25/19~ C temperature and 70/80% relative humidity. Plants were irrigated on alternate days with a Long Ashton nutrient solution containing 10 mM NO~ as nitrogen source (Hewitt 1966; de la Haba et al. 1988). On the eighth day, seedlings of uniform size were removed, their roots rinsed in distilled water and transferred to glass beakers containing 450 ml of the above nutrient solution but with 0.5 mM KNO3. After 6 d, the solution was replaced by a nitrogen-free nutrient solution (Hewitt 1966). For induction of NO~-- and NO~-uptake systems, plants starved of nitrogen for 6 d were pretreated for 24 h with a solution consisting of 0.1 mM KNO3 and 1 mM CaSO4, In the experiments relating to Fig. I, some of the plants were pretreated with 0.1 mM KNO2 instead of KNO3. Measurements of NO~ and NO~ uptake. Pretreated intact plants were transferred to either NO~ o r N O 2 solution to assess uptake. Unless stated otherwise, NO~- and NO 2 uptake solutions (250 ml) consisted of 10 mM 2-(N-morpholine)ethanesulfonic acid (Mes)KOH buffer, pH 5.5, 1 mM CaSO4 and either 0.1 mM KNO3 or 0A mM KNO2. The solutions were continuously bubbled with air. The net uptake of NO~- or NO~- by sunflower plants was estimated by measuring ion depletion from the external solution under light (200 lamol-m-Z.s-1) and 25~ C. Rates of uptake were constant (i.e. independent of the remaining external ion concentration) during the routine uptake assay time (2-3 h). It should be emphasized that, in plants prctreated and assayed with NO 3- concentrations close to those used in this study, net NO~- uptake was found to be practically identical to NO~- influx (Jackson et al. 1976; Ingemarsson et al. 1987). Uptake data were expressed on the basis of one gram of fresh weight of roots.
E. Agiiera et al. : Nitrate and nitrite uptake by sunflower (4 ml.g -1 FW) consisting of 0.1 M 2-amino-2-(hydroxymethyl)1,3-propanediol (Tris)-HC1, pH 7.5, 10 mM cysteine, 1 mM EDTA-, and 5 p.M flavine-adenine dinucleotide (FAD). The homogenate was centrifuged at 25000.g for 25 min at 4~ C and the supernatant was used for determination of NiR activity, as described by Losada and Paneque (1971). The reaction mixture contained 75 mM TrisHC1 buffer, pH 7.5, 2 mM NaNO2, 0.75 mM methyl viologen, 21 mM Na2S204 (dissolved in 0.3 M NaHCO3), and the adequate amount of extract. Activities of NR and NiR were expressed as ~tmol NO~ formed or reduced, respectively, h-1.g-1 root FW.
Analytical determinations. Solution NO~- concentrations were determined spectrophotometrically at 210 nm in acid solution as described by Cawse (1967). The NO~ concentrations, both in uptake solutions and NiR assay mixtures, were measured colorimetrically by the diazotization method of Snell and Snell (| 949). The data presented are from one representative experiment of at least three independent experiments performed on different plant cultures. Each determination was made in triplicate.
Results Induction o f N O ~ - and N O z - u p t a k e systems. N i t r a t e c o n c e n t r a t i o n in sunflower roots before n i t r o g e n - s t a r v a tion r a n g e d between 2.7 a n d 3.1 ~tmol.g -~ F W . After the 6-d s t a r v a t i o n period, e n d o g e n o u s N O ~ c o n t e n t became imperceptible. Nitrite was never detected in r o o t tissue. Figure 1 A shows that n i t r o g e n - d e p r i v e d p l a n t s could n o t initially take u p NO~- w h e n exposed to the N O ~ solution. After 4 h, however, plants developed the ability to take u p NO~-. W h e n the n i t r o g e n - s t a r v e d
~-
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Kinetics of NO 3 and NO Z uptake, Kinetic parameters of enzymecatalyzed reactions can be easily and accurately determined by analyzing substrate-depletion curves by the integrated form of Michaelis-Menten equation (Orsi and Tipton 1979). This method has been successfully used to calculate kinetic data of NO 2 uptake and reduction by the microalga Chlamydomonas reinhardtii (C6rdoba et al, 1986). The Km values for NO~ and NO~- uptake by intact sunflower plants were therefore calculated from NO3-and NO,--depletion curves by using the integrated rate equation rearranged in the Hanes-Woolf form to give a linear plot (Orsi and Tipton 1979):
~-
t 1 (So-S) Km In (So/S)- Vm,x In (So/S) -~ Vmax
~
8
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i
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(h)
B ,,~ 16
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e/o
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8
where t is time in h, So is the initial concentration of NO 3 or NO2, S is the concentration of NO~- or NO 2 at the time t, Km is the Michaelis constant and Vmaxis the maximum uptake rate.
Assays of NR and NiR. Because of the high instability of sunflower NR after extraction, even in the presence of protectants (Agtiera et al. 1987b), NR activity was determined by the vacuum-infiltration in-vivo assay described by Maurifio et al. (1986). The assay mixture consisted of 0.1 M K-phosphate buffer, pH 7.5, 50 mM KNOa, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1% (v/v) n-propanol. Nitrite reductase was extracted from roots by macerating the tissue, in a chilled mortar, with sand and an extraction medium
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Fig. 1 A, B. Effects of NO~ and NO~ pretreatments on the uptake of NO~- (A) and NO~- (B) by intact sunflower plants. Prior to uptake experiments, nitrogen-starved plants were either not pretreated (0) or pretreated for 24 h with 0.1 mM NO~ (*) or 0.1 mM NO 2 (nn).Initial concentrations of NO 3 and NO~ were 0.5 mM
E. Agfieraet al. : Nitrate and nitrite uptake by sunflower
151
Table 1. Effects of cycloheximide and sulfhydryl-group reagents on the uptake and reduction of NO~ and NO 2 by intact sunflower plants. Plants were either pretreated or not with CHI (10 gg. m l - 1) for 24 h or with p H M B (0.5 mM) or N E M (0.3 mM) for 1 h. Afterwards, plants were transferred to the NO~-uptake and N O z - u ptake solutions. Uptake occurred for 6 h after which time N R and NiR activities were determined in the roots Inhibitor pretreatment
NO~ uptake experiment
NO 2 uptake experiment
.%
200
A
,-s
160 = o
120 0= ~: o ~"
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Z
NO~ uptake rate
NR NO2 activity uptakerate
NiR activity
o
;,
g m o l . h - 1.g -1 FW None CHI pHMB NEM
1.64 ND" 1.48 ND
0.37 ND 0.26 ND
8
1'o
Time (hi
0.96 0.04 0.84 0.36
31.7 3.4 32.7 35.5
" N D = not detected
plants were pretreated with 0.1 mM NO~ for 24 h and subsequently transferred to the uptake solution, NO 3 uptake proceeded linearly from the beginning. Nitrite uptake by nitrogen-deprived sunflower plants also showed a lag phase upon first exposure to the NO 2 solution (Fig. 1 B). Pretreatment of the plants with 0.1 mM NO~- or NOz prior exposure to NOz also resulted in high initial rates of NO 2 uptake. Nitrate was more effective than NO 2 in the induction of ability to take up NO2. The presence of the protein-synthesis inhibitor cycloheximide (CHI) during NOa-pretreatment drastically prevented the subsequent uptake of NO~ and NO2 (Table 1), indicating that the development of both uptake systems involved de-novo protein synthesis. Cycloheximide also negatively affected the synthesis of NR and NiR in the roots (Table 1). Effects of sulfhydryl-group reagents on the uptake and reduction of NO~ and NO 2. There is evidence that sulfhydryl groups are often involved in carrier-mediated ion transport (Kochian and Lucas 1982). Hence, the effects of the sulfhydryl-group reagents p-hydroxymercuribenzoate (pHMB) and N-ethylmaleimide (NEM) on the uptake of NO 3 and NO 2 were tested. Results show (Table 1) that inclusion of 0.5 mM pHMB for 1 h, prior to assay, had little effect on the subsequent uptake of NO~ and NO 2. In contrast, pretreatment with 0.3 mM NEM greatly inhibited both NO~ and NO 2 uptake. It was also observed that root NR activity showed a higher sensitivity in vivo to NEM than to pHMB. However, NiR activity was unaffected by the above compounds (Table 1). Kinetics of NO 3 and NO 2 uptake. Figure 2A shows a typical progress curve of NO 3 depletion in a solution initially containing 0.2 mM NO3. From this curve, rates of NO 3 uptake at various external NO~- concentrations were estimated using a tangential method. Plot of the rates versus NO~ concentrations exhibited Michaelis-
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160
So-S / tn (So/S) Fig. 2. A Time course of NO~- depletion of external solution by intact sunflower plants. B Plot of the rates of NO~ uptake versus NO 3 concentration in the medium. The rates of NO 3 uptake were calculated by drawing a series of tangents to the NO,-depletion curve in A at points corresponding to different NO~ concentrations. C Hanes-Woolf representation of the integrated form of Michaelis-Menten equation for NO~ uptake. The NO3-depletion curve (A) was analyzed by the integrated form of the MichaelisMenten equation, as described in Material and methods; So and S are expressed in gM and time (t) in h
Menten saturation kinetics (Fig. 2 B) with a K mof 45 gM (Fig. 2C), indicating that NO 3 uptake by sunflower plants is a carrier-mediated process. The depletion of NO z from uptake solution by sunflower plants is shown in Fig. 3 A. Uptake also exhibited saturation kinetics (Fig. 3 B). When the data of the NO~-depletion curve were analyzed by the integrated Michaelis-Menten equation, a Km value for NO~ of 23 ~tM was calculated (Fig. 3 C). Effect on anaerobiosis on the uptake and reduction of NO~ and NO2. External solutions were always bubbled
152
E. Agfiera et al. : Nitrate and nitrite uptake by sunflower
0.20
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9
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Fig. 4A, B. Influence of pH on NO~ uptake and reduction (A) and on NO 2 uptake and reduction (B) in sunflower plants. Rates of NO~- and NO 2 uptake were measured in media buffered with 5mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes)-5 mM Mes adjusted at the indicated pH values. The NR and NiR activities in roots were determined in assay media buffered with 75 mM citrate-3-(N-morpholino)propanesulfonic acid (Mops)-glyeine buffer at the indicated pH values
o
i
." 2
/ -20
0
' 20
' 40
6'0
' 80
' 100
So-S / tn {S0/Sl Fig, 3. A Time course of NO 2- depletion of external solution by intact sunflower plants. B Plot of the rates of NO~- uptake versus NO~- concentration in the medium. C Hanes-Woolf representation of the integrated form of Michaelis-Menten equation for NO~ uptake. Experimental procedures were as in Fig. 2, except that NO 2 uptake was assayed
2. Effect of anaerobiosis on the uptake and reduction of NO~- and NO 2 by intact sunflower plants. Uptake solutions were bubbled either with air or with oxygen-free nitrogen gas. After a 6-h uptake period, NR and NiR activities were determined in the roots Table
Treatment
NO~- uptake experiment NO~ uptake rate
NO~ uptake experiment NR activity
NO 2 uptake rate
NiR activity
0.96 0.16
26.8 29.8
with air in order to maintain an adequate oxygen tension near the roots. When the solutions were made nearly anaerobic by continuous bubbling with N 2 instead of air, N O ~ uptake was fully abolished while N O ~ uptake greatly diminished (Table 2). This observation indicates that both NO~-- and N O 2 - u p t a k e systems are highly dependent on a continuous supply of metabolic energy. The removal of oxygen during the 6-h uptake period assayed did not substantially affect N R and N i R activities in the roots (Table 2).
Effect of p H on the uptake and reduction of NO 3 and NO2. Uptake and reduction processes of N O 3 and N O 2 were differently influenced by p H (Fig. 4). Uptake of both anions was higher at a mildly acid pH, maximal rates occurring at an external p H value around 5.5. In contrast, reduction of N O ~ and N O ] exhibited optimal pHs of 7.5 and 8.0, respectively.
g m o l - h - 1.g -1 F W Aerobiosis Anaerobiosis
1.24 ND a
" N D = n o t detected
0.20 0.17
Discussion
The development of accelerated N O S uptake following initial exposure to N O ~ has been observed in some
E. Agiiera et al. : Nitrate and nitrite uptake by sunflower plants (Tompkins et al. 1978; Deane-Drummond 1984; MacKown and McClure 1988). Nitrate uptake into nitrogen-starved sunflower plants also showed a lag phase (Fig. 1 A). Nevertheless, uptake was high and linear from the beginning when the plants were pretreated with NO 3 (Fig. 1 B). On the other hand, the presence of CHI during NO;- pretreatment prevented NO 3 uptake (Table 1), indicating that NO 3 induces de-novo synthesis of the NO;--uptake system, as previously found in other plants (Tompkins et al. 1978; Deane-Drummond 1984; Morgan etal. 1985b; MacKown and McClure 1988). We did not detect a low-capacity NO~-uptake system in nitrogen-starved sunflower plants comparable to that found in barley roots (Behi et al. 1988). The kinetics of NO 3 uptake by sunflower plants (Fig. 2) is consistent with a carrier-mediated mechanism with negligible passive uptake. This is corroborated by the complete suppression of NO 3 uptake when the plants were pretreated with CHI or NEM (Table 1). For the NO 3 concentration range used in this work (up to 0.2 mM), only a single uptake system with a Km of 45 gM was detected (Fig. 2). This value is within the range of Km values found in other higher plants either for single uptake system or for the mechanism I (high affinity) of dual systems (Beevers and Hageman 1983; Goyal and Huffaker 1986). In those uptake systems where both carriermediated and free-diffusion components were involved, CHI and sulfhydryl-group reagents abolished the former without affecting the latter (Serra et al. 1978; Kochian and Lucas 1982; Ibarlucea et al. 1983). The NO 3-uptake system of sunflower plants exhibited a lower sensitivity to pHMB than to NEM (Table 1). A similar response has been found for the NO~--uptake system of Chlamydomonas reinhardtii (C6rdoba et al. 1986) and sunflower (this paper). N-ethylmaleimide is known to permeate membranes more readily than pHMB (Kochian and Lucas 1982; C6rdoba et al. 1986). This agrees with the observation (Table 1) that NR, which contains SH-groups that are essential for its catalytic activity (Guerrero et al. 1981; Beevers and Hageman 1983), was inactivated in vivo in a greater extent by NEM than by pHMB. Sunflower plants were also able to develop a NOzuptake system when exposed to NO 3 or NO 2 (Fig. 1 B). Nitrite uptake was greatly prevented by CHI (Table 1), indicating that induction of the NO~-uptake system also involved de-novo protein synthesis. The existence of an inducible NO~-uptake system, scarcely studied in higher plants, has only been observed in wheat (Jackson et al. 1974; Goyal and Huffaker 1986) and barley (Ibarlucea et al. 1983). Nitrite uptake by intact sunflower plants exhibited Michaelis-Menten kinetics (Fig. 3), indicating a carrier-mediated mechanism. It was more susceptible to inactivation by NEM than by pHMB, as found for NO 3 uptake (Table 1) and also for the NOz-uptake system of the green alga Chlamydomonas reinhardtii (C6rdoba et al. 1986). Nevertheless, in contrast to NR, NiR from sunflower roots was not inactivated in vivo by NEM (Table 1), which agrees with the lower sensitivity of NiR to sullhydryl-group reagents found in vitro (Beevers and Hageman 1983). In contrast to NO;- uptake, NO~- uptake was not fully suppressed by pretreat-
153 ments with either CHI or NEM (Table 1), or when the uptake solution was made anaerobic (Table 2). This might indicate that some NO2 could enter into the root cells by a passive non-mediated mechanism. The observation that NO~ and NO~ uptake was drastically abolished when oxygen was removed from the uptake solution (Table 2) indicates that uptake is highly dependent on metabolic energy derived from root respiration. Ullrich (1987) has postulated that NO 3 is taken up by a 2H+/NO;- symport coupled to an ATPdependent proton-extrusion pump which generates the proton electrochemical gradient required for NO~ transport against a concentration gradient. We found that not only NO 3 uptake but also NO 2 uptake proceeded faster at acid pH, drastically decreasing at external pH values above 5.5 (Fig. 4A). According to the proposed 2H+/NO3 symport machanism (Ullrich 1987), which might be extensible to NO2 uptake, a high proton concentration in the external medium would build up the proton electrochemical gradient across the plasma membrane and, therefore, the driving force both for NO3 and NO~- uptake. Reduction of NO 3 and NO 2 exhibited different responses to pH, showing optimum pH values of 7.5 and 8.0, respectively (Fig. 4B). The results presented in this paper also show that in sunflower plants uptake and reduction processes, both of NO 3 and NO 2, exhibit different characteristics. First, the observed Km values for uptake are lower than the reported Km values for reduction (Guerrero et al. 1981 ; Beevers and Hageman 1983). Second, uptake and reduction proceed at different rates. Nitrate-uptake rates are four to six times higher than NO3-reduction rates in the roots. This indicates that a portion of NO 3 absorbed by the plant is accumulated in the root and- or translocated to the shoot. In corn roots taking up 15NOj after induction of NO 3-uptake system and NR activity, about 30% of the incoming 15NO 3 was reduced, 20% accumulated and 50% translocated (Morgan et al. 1985a). In contrast to NO 3 uptake, NOz-uptake rates are about 30 times lower than NO~-reduction rates in the roots, indicating that all the NO~ taken up by sunflower plants is reduced in the roots. In fact, NO2 was never detected in the root tissue. Third, anaerobic conditions greatly suppress NO;- and NO 2 uptake without substantially affecting their reduction. And fourth, uptake and reduction processes show different pH optima. This research was supported by grant PB86-0232 from the Direcci6n General de Investigaci6nCientificay T6cnica (Spain). One of us (E.A.) thanks the Consejeria de Educaci6n y Ciencia de la Junta de Andalucia for the tenure of a fellowship.We thank Miss G. Alcalfi and Miss C. Santos for their valuable technical and secretarial assistance. References Agiiera, E., de la Haba, P., Maldonado, J.M. (1987a) Induction of nitrate reductase, nitrite reductase and glutaminesynthetase by nitrate and/or nitrite in germinating sunflower cotyledons. Effect of plant hormones. Plant Physiol. (Life Sci. Adv.) 6, 255-258 Agfiera, E., de la Haba, P., Maldonado, J.M. (1987b) In vitro stabilization and tissue distributionof nitrogen-assimilatingenzymes in sunflower.J. Plant Physiol. 128, 443-449
154 Aslam, M., Huffaker, R.C. (1989) Role of nitrate and nitrite in the induction of nitrite reductase in leaves of barley seedlings. Plant Physiol. 91, 1152-1156 Bagchi, S.N., Rai, U.N., Rai, A.N., Singh, H.N. (1985) Nitrate metabolism in the cyanobacterium Anabaena cycadeae: Regulation of nitrate uptake and reductase by ammonia. Physiol. Plant. 63, 322-326 Beevers, L., Hageman, R.H. (1983) Uptake and reduction of nitrate: bacteria and higher plants. In: Encyclopedia of plant physiology, N.S., vol. 15A: Inorganic plant nutrition, pp. 351375, Lfiuchli, A., Bieleski, R.L., eds. Springer, Berlin Heidelberg New York Behl, R., Tischner, R., Raschke, K. (1988) Induction of a highcapacity nitrate-uptake mechanism in barley roots prompted by nitrate uptake through a constitutive low-capacity mechanism. Planta 176, 235-240 Breteler, H., Luczak, E. (1982) Utilization of nitrite and nitrate by dwarf bean. Planta 156, 226-232 Cawse, P.A. (1967) The determination of nitrate in soil solutions by ultraviolet spectrophotometry. Analyst 92, 311-315 C6rdoba, F., Cfirdenas, J., Fernfindez, E. (1986) Kinetic characterization of nitrite uptake and reduction by Chlamydomonas reinhardtii. Plant Physiol. 82, 904-908 Cresswell, R.C., Syrett, P.J. (1981) Uptake of nitrate by the diatom Phaeodactylum tricornutum. J. Exp. Bot. 32, 19-25 De la Haba, P., Agfiera, E., Maldonado, J.M. (1988) Development of nitrogen-assimilating enzymes in sunflower cotyledons during germination as affected by the exogenous nitrogen source. Planta 173, 52-57 Deane-Drummond, C.E. (1984) The apparent induction of nitrate uptake by Chara corallina cells following pretreatment with or without nitrate and chlorate. J. Exp. Bot. 35, 1182-1193 Dhugga, K.S., Waines, J.G., Leonard, R.T. (1988) Correlated induction of nitrate uptake and membrane polypeptides in corn roots. Plant Physiol. 87, 120-125 Goyal, S.S., Huffaker, R.C. (1986) The uptake of NO~-, NO~-, and NH,~ by intact wheat (Triticum aestivum) seedlings. Plant Physiol. 82, 1051-1056 Guerrero, M.G., Vega, J.M., Losada, M. (1981) The assimilatory nitrate-reducing system and its regulation. Annu. Rev. Plant Physiol. 32, 169-204 Hewitt, E.J. (1966) Sand and water culture methods used in the study of plant nutrition, 2nd rev. edn. Commonwealth Bureau of Horticultural and Plantation Crops, East Malling Tech. Commun. No. 22 Ibarlucea, J.M., Llama, M.J., Serra, J.L., Macarulla, J.M. (1983) Mixed-transfer kinetics of nitrite uptake in barley (Hordeum vulgare L. cv. Miranda) seedlings. Plant Sci. Lett. 29, 339-347 Ingemarsson, B., Oscarson, P., af Ugglas, M., Larsson, C.-M. (1987) Nitrogen utilization in Lemna. II. Studies of nitrate uptake using 1 3 N 0 3. Plant Physiol. 85, 860-864
E. Agfiera et al. : Nitrate and nitrite uptake by sunflower Jackson, W.A., Johnson, R.E., Volk, R.J. (1974) Nitrite uptake patterns in wheat seedlings as influenced by nitrate and ammonium. Physiol. Plant. 32, 108-114 Jackson, W.A., Kwik, K.D., Volk, R.J., Butz, R.G. (1976) Nitrate influx and efflux by intact wheat seedlings: effects of prior nitrate nutrition. Planta 132, 149-156 Kochian, UV., Lucas, W.J. (1982) Potassium transport in corn roots. I. Resolution of kinetics into a saturable and linear component. Plant Physiol. 70, 1723-1731 Lea, P.J., Miflin, B.J. (1979) Photosynthetic ammonia assimilation. In: Encyclopedia of plant physiology, N.S., voL 6: Photosynthesis II, pp. 445-456, Gibbs, M., Latzko, E., eds. Springer, Berlin Heidelberg New York Losada, M., Paneque, A. (1971) Nitrite reductase. Methods Enzymol. 23, 487-491 McClure, P.R., Omholt, T.E., Pace, G.M., Bouthyette, P.Y. (1987) Nitrate-induced changes in protein synthesis and translation of RNA in maize roots. Plant Physiol. 84, 52-57 MacKown, C.T., McClure, P.R. (1988) Development of accelerated net nitrate uptake. Effects of nitrate concentration and exposure time. Plant Physiol. 87, 162-166 Maduefio, F., Vega-Palas, M.A., Flores, E., Herrero, A. (1988) A cytoplasmic-membrane protein repressible by ammonium in Synechococcus R2: altered expression in nitrate-assimilation mutants. FEBS Lett. 239, 289-291 Maurifio, S.G., Echevarrla, C., Mejias, J.A., Vargas, M.A., Maldonado, J.M. (1986) Properties of the in vivo nitrate reductase assay in maize, soybean and spinach leaves. J. Plant Physiol. 124, 123-130 Morgan, M.A., Jackson, W.A., Volk, R.J. (1985a) Uptake and assimilation of nitrate by corn roots during and after induction of the nitrate uptake system. J. Exp. Bot. 36, 859-869 Morgan, M.A., Volk, R.J., Jackson, W.A. (1985b)p-Fluorophenylalanine-induced restriction of ion uptake and assimilation by maize roots. Plant Physiol. 77, 718 721 Orsi, B.A., Tipton, K.F. (1979) Kinetic analysis of progress curves. Methods Enzymol. 63, 159 183 Serra, J.L., Llama, M.J., Cadenas, E. (1978) Nitrate utilization by the diatom Skeletonema costatum. I. Kinetics of nitrate uptake. Plant Physiol. 62, 987-990 Snell, F.D., Snell, C.T. (1949) Colorimetric methods of analysis, vol. 2. Van Nostrand, New York Tompkins, G.A., Jackson, W.A., Volk, R.J. (1978) Accelerated nitrate uptake in wheat seedlings: effects of ammonium and nitrite pretreatments and of 6-methylpurine and puromycin. Physiol. Plant. 43, 166-171 Ullrich, W.R. (1987) Nitrate and ammonium uptake in green algae and higher plants: mechanism and relationship with nitrate metabolism. In: Inorganic nitrogen metabolism, pp. 32-38, Ullrich, W.R., Aparicio, P.J., Syrett, P.J., Castillo, F., eds. Springer, Berlin Heidelberg New York
Planta (1990)182:149-154
9 Springer-Verlag1990
Nitrate and nitrite uptake and reduction by intact sunflower plants E. Agiiera, P. de la Haba, A.G. Fontes, and J.M. Maldonado* Departamento de BiologiaVegetaly Ecologia, Divisi6n de FisiologiaVegetal, Facultadde Ciencias,Universidadde C6rdoba, E-14004 C6rdoba, Spain Received 3 February; accepted 6 April 1990
Abstract. Nitrogen-starved sunflower plants (Helianthus annuus L. cv. Peredovic) cannot absorb NO 3 or NO~ upon initial exposure to these anions. Ability of the plants to take up NO 3 and NO~ at high rates from the beginning was induced by a pretreatment with NO 3 . Nitrite also acted as inducer of the NO~-uptake system. The presence of cycloheximide during NOf-pretreatment prevented the subsequent uptake of NO 3 and NO2, indicating that both uptake systems are synthesized de novo when plants are exposed to NOr. Cycloheximide also suppressed nitrate-reductase (EC 1.6.6.1) and nitrite-reductase (EC 1.7.7.1) activities in the roots. The sulfhydryl-group reagent N-ethylmaleimide greatly inhibited the uptake of NO 3 and NO 2. Likewise, Nethylmaleimide promoted in vivo the inactivation of nitrate reductase without affecting nitrite-reductase activity. Rates of NO 3 and NO~- uptake as a function of external anion concentration exhibited saturation kinetics. The calculated Km values for NO 3 and N O f uptake were 45 and 23 ~tM, respectively. Rates of NO~ uptake were four to six times higher than NOs-reduction rates in roots. In contrast, NOz-uptake rates, found to be very similar to NOs-uptake rates, were much lower (about 30 times) than NOz-reduction rates. Removal of oxygen from the external solution drastically suppressed NO 3 and NO~- uptake without affecting their reduction. Uptake and reduction were also differentially affected by pH. The results demonstrate that uptake of NO 3 and NO 2 into sunflower plants is mediated by energy-dependent inducible-transport systems distinguishable from the respective enzymatic reducing systems. Key words: Helianthus- Nitrate (reduction, u p t a k e ) Nitrite (reduction, uptake)
Introduction The utilization of inorganic nitrogen, either NO 3 or NO~, by higher plants involves the following steps: up* To whom correspondenceshould be addressed Abbreviations: CHI=cycloheximide; NEM=N-ethylmaleimide; NiR=nitrite reductase; NR=nitrate reductase; pHMB=phydroxymercuribenzoate
take of NO 3 or NO~ into the plant, their reduction to NH +, and incorporation of NH + to carbon skeletons to produce amino acids. Reduction of NO 3 to NH~proceeds by the sequential action of the enzymes nitrate reductase (NR) and nitrite reductase (NiR) (Guerrero et al. 1981). Ammonium is then assimilated mostly through the glutamine synthetase/glutamate synthase cycle (Lea and Miflin 1979). In various plants, the presence of NO 3 in the medium induces the development of a NOs-uptake system (Tompkins et al. 1978; Deane-Drummond 1984; MacKown and McClure 1988). A constitutive low-capacity uptake system for NO 3 has been found to be additionally present in barley plants which have not been previously exposed to NO 3 (Behl et al. 1988). In the diatom Phaeodactylum trieornutum (Cresswell and Syrett 1981) and in the cyanobacterium Anabaena cycadeae (Bagchi et al. 1985), the NO~--uptake system appears to be NH~-repressible rather than NOs-inducible. Recently, a protein of about 48 kDa has been detected in the plasma membrane of the cyanobacterium Synechococcus R2 grown on NO3, but not in NH~-grown cells (Maduefio et al. 1988). Likewise, the presence of NO 3 in the medium has been shown to induce the synthesis of 30- and 40-kDa proteins in the plasma membrane (Dhugga et al. 1988) and tonoplast (McClure et al. 1987) in corn roots. From these observations it has been suggested that the NO3-inducible membrane proteins might have a role in the uptake of NO3. Unlike NO 3 uptake, NO~ uptake by higher plants has been scarcely studied. The occurrence of a NO zuptake system has been only demonstrated in wheat (Jackson et al. 1974; Goyal and Huffaker 1986), dwarf bean (Breteler and Luczak 1982) and barley (Ibarlucea et al. 1983) seedlings. In sunflower plants, NR is specifically induced by NO 3 while NiR synthesis is enhanced both by NO 3 and NO z (Agfiera et al. 1987a; de la Haba et al. 1988). Aslam and Huffaker (1989) have recently reported that in barley leaves NiR is induced by NO 3 directly, i.e. without being reduced to NO 2, and that absorbed NO 2 induces the enzyme activity indirectly after being oxidized to NO 3 within the leaf. The present study was undertaken to investigate the existence of NO 3- and NOz-uptake systems in intact sunflower plants. As found in other plants, NO 3- and NO2-uptake systems
150 were s u b s t r a t e - i n d u c i b l e . Both u p t a k e systems have been characterized, s h o w i n g different properties f r o m those exhibited b y the respective r e d u c i n g systems.
Material and methods Plant culture. Helianthus annuus L. cv. Peredovic (supplied by Eurosemillas, C6rdoba, Spain) was germinated and grown on a 1:1 (v/v) mixture of perlite and vermiculite in a growth chamber with 16 h of light daily (200 ~mol. m - 2. s- x of photosynthetically active radiation) and a day/night regime of 25/19~ C temperature and 70/80% relative humidity. Plants were irrigated on alternate days with a Long Ashton nutrient solution containing 10 mM NO~ as nitrogen source (Hewitt 1966; de la Haba et al. 1988). On the eighth day, seedlings of uniform size were removed, their roots rinsed in distilled water and transferred to glass beakers containing 450 ml of the above nutrient solution but with 0.5 mM KNO3. After 6 d, the solution was replaced by a nitrogen-free nutrient solution (Hewitt 1966). For induction of NO~-- and NO~-uptake systems, plants starved of nitrogen for 6 d were pretreated for 24 h with a solution consisting of 0.1 mM KNO3 and 1 mM CaSO4, In the experiments relating to Fig. I, some of the plants were pretreated with 0.1 mM KNO2 instead of KNO3. Measurements of NO~ and NO~ uptake. Pretreated intact plants were transferred to either NO~ o r N O 2 solution to assess uptake. Unless stated otherwise, NO~- and NO 2 uptake solutions (250 ml) consisted of 10 mM 2-(N-morpholine)ethanesulfonic acid (Mes)KOH buffer, pH 5.5, 1 mM CaSO4 and either 0.1 mM KNO3 or 0A mM KNO2. The solutions were continuously bubbled with air. The net uptake of NO~- or NO~- by sunflower plants was estimated by measuring ion depletion from the external solution under light (200 lamol-m-Z.s-1) and 25~ C. Rates of uptake were constant (i.e. independent of the remaining external ion concentration) during the routine uptake assay time (2-3 h). It should be emphasized that, in plants prctreated and assayed with NO 3- concentrations close to those used in this study, net NO~- uptake was found to be practically identical to NO~- influx (Jackson et al. 1976; Ingemarsson et al. 1987). Uptake data were expressed on the basis of one gram of fresh weight of roots.
E. Agiiera et al. : Nitrate and nitrite uptake by sunflower (4 ml.g -1 FW) consisting of 0.1 M 2-amino-2-(hydroxymethyl)1,3-propanediol (Tris)-HC1, pH 7.5, 10 mM cysteine, 1 mM EDTA-, and 5 p.M flavine-adenine dinucleotide (FAD). The homogenate was centrifuged at 25000.g for 25 min at 4~ C and the supernatant was used for determination of NiR activity, as described by Losada and Paneque (1971). The reaction mixture contained 75 mM TrisHC1 buffer, pH 7.5, 2 mM NaNO2, 0.75 mM methyl viologen, 21 mM Na2S204 (dissolved in 0.3 M NaHCO3), and the adequate amount of extract. Activities of NR and NiR were expressed as ~tmol NO~ formed or reduced, respectively, h-1.g-1 root FW.
Analytical determinations. Solution NO~- concentrations were determined spectrophotometrically at 210 nm in acid solution as described by Cawse (1967). The NO~ concentrations, both in uptake solutions and NiR assay mixtures, were measured colorimetrically by the diazotization method of Snell and Snell (| 949). The data presented are from one representative experiment of at least three independent experiments performed on different plant cultures. Each determination was made in triplicate.
Results Induction o f N O ~ - and N O z - u p t a k e systems. N i t r a t e c o n c e n t r a t i o n in sunflower roots before n i t r o g e n - s t a r v a tion r a n g e d between 2.7 a n d 3.1 ~tmol.g -~ F W . After the 6-d s t a r v a t i o n period, e n d o g e n o u s N O ~ c o n t e n t became imperceptible. Nitrite was never detected in r o o t tissue. Figure 1 A shows that n i t r o g e n - d e p r i v e d p l a n t s could n o t initially take u p NO~- w h e n exposed to the N O ~ solution. After 4 h, however, plants developed the ability to take u p NO~-. W h e n the n i t r o g e n - s t a r v e d
~-
"7
o
/
Kinetics of NO 3 and NO Z uptake, Kinetic parameters of enzymecatalyzed reactions can be easily and accurately determined by analyzing substrate-depletion curves by the integrated form of Michaelis-Menten equation (Orsi and Tipton 1979). This method has been successfully used to calculate kinetic data of NO 2 uptake and reduction by the microalga Chlamydomonas reinhardtii (C6rdoba et al, 1986). The Km values for NO~ and NO~- uptake by intact sunflower plants were therefore calculated from NO3-and NO,--depletion curves by using the integrated rate equation rearranged in the Hanes-Woolf form to give a linear plot (Orsi and Tipton 1979):
~-
t 1 (So-S) Km In (So/S)- Vm,x In (So/S) -~ Vmax
~
8
9/ o
/
9 i
0
t/ 9
t
/o
o/
z
~o__o__O--O ~ o / i
i
i
0
2
4 Time
0/~ 6
i
8
(h)
B ,,~ 16
C:n
e/o
12
8
where t is time in h, So is the initial concentration of NO 3 or NO2, S is the concentration of NO~- or NO 2 at the time t, Km is the Michaelis constant and Vmaxis the maximum uptake rate.
Assays of NR and NiR. Because of the high instability of sunflower NR after extraction, even in the presence of protectants (Agtiera et al. 1987b), NR activity was determined by the vacuum-infiltration in-vivo assay described by Maurifio et al. (1986). The assay mixture consisted of 0.1 M K-phosphate buffer, pH 7.5, 50 mM KNOa, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1% (v/v) n-propanol. Nitrite reductase was extracted from roots by macerating the tissue, in a chilled mortar, with sand and an extraction medium
2z~
z
o~"
./
0 i
t
i
t
0
2
~
6
Time (h)
Fig. 1 A, B. Effects of NO~ and NO~ pretreatments on the uptake of NO~- (A) and NO~- (B) by intact sunflower plants. Prior to uptake experiments, nitrogen-starved plants were either not pretreated (0) or pretreated for 24 h with 0.1 mM NO~ (*) or 0.1 mM NO 2 (nn).Initial concentrations of NO 3 and NO~ were 0.5 mM
E. Agfieraet al. : Nitrate and nitrite uptake by sunflower
151
Table 1. Effects of cycloheximide and sulfhydryl-group reagents on the uptake and reduction of NO~ and NO 2 by intact sunflower plants. Plants were either pretreated or not with CHI (10 gg. m l - 1) for 24 h or with p H M B (0.5 mM) or N E M (0.3 mM) for 1 h. Afterwards, plants were transferred to the NO~-uptake and N O z - u ptake solutions. Uptake occurred for 6 h after which time N R and NiR activities were determined in the roots Inhibitor pretreatment
NO~ uptake experiment
NO 2 uptake experiment
.%
200
A
,-s
160 = o
120 0= ~: o ~"
BO t.O
Z
NO~ uptake rate
NR NO2 activity uptakerate
NiR activity
o
;,
g m o l . h - 1.g -1 FW None CHI pHMB NEM
1.64 ND" 1.48 ND
0.37 ND 0.26 ND
8
1'o
Time (hi
0.96 0.04 0.84 0.36
31.7 3.4 32.7 35.5
" N D = not detected
plants were pretreated with 0.1 mM NO~ for 24 h and subsequently transferred to the uptake solution, NO 3 uptake proceeded linearly from the beginning. Nitrite uptake by nitrogen-deprived sunflower plants also showed a lag phase upon first exposure to the NO 2 solution (Fig. 1 B). Pretreatment of the plants with 0.1 mM NO~- or NOz prior exposure to NOz also resulted in high initial rates of NO 2 uptake. Nitrate was more effective than NO 2 in the induction of ability to take up NO2. The presence of the protein-synthesis inhibitor cycloheximide (CHI) during NOa-pretreatment drastically prevented the subsequent uptake of NO~ and NO2 (Table 1), indicating that the development of both uptake systems involved de-novo protein synthesis. Cycloheximide also negatively affected the synthesis of NR and NiR in the roots (Table 1). Effects of sulfhydryl-group reagents on the uptake and reduction of NO~ and NO 2. There is evidence that sulfhydryl groups are often involved in carrier-mediated ion transport (Kochian and Lucas 1982). Hence, the effects of the sulfhydryl-group reagents p-hydroxymercuribenzoate (pHMB) and N-ethylmaleimide (NEM) on the uptake of NO 3 and NO 2 were tested. Results show (Table 1) that inclusion of 0.5 mM pHMB for 1 h, prior to assay, had little effect on the subsequent uptake of NO~ and NO 2. In contrast, pretreatment with 0.3 mM NEM greatly inhibited both NO~ and NO 2 uptake. It was also observed that root NR activity showed a higher sensitivity in vivo to NEM than to pHMB. However, NiR activity was unaffected by the above compounds (Table 1). Kinetics of NO 3 and NO 2 uptake. Figure 2A shows a typical progress curve of NO 3 depletion in a solution initially containing 0.2 mM NO3. From this curve, rates of NO 3 uptake at various external NO~- concentrations were estimated using a tangential method. Plot of the rates versus NO~ concentrations exhibited Michaelis-
B
U-
,~ 2.~ i 0
E ._~ 1.6
n~
o.8
z
0 0
f
I
20
60
r
I
100+
14.0
NO~ concenfrai'ion (t~M) 6 \ o
4..
2 i
-t+O
0
40
i
80
I
120
I
160
So-S / tn (So/S) Fig. 2. A Time course of NO~- depletion of external solution by intact sunflower plants. B Plot of the rates of NO~ uptake versus NO 3 concentration in the medium. The rates of NO 3 uptake were calculated by drawing a series of tangents to the NO,-depletion curve in A at points corresponding to different NO~ concentrations. C Hanes-Woolf representation of the integrated form of Michaelis-Menten equation for NO~ uptake. The NO3-depletion curve (A) was analyzed by the integrated form of the MichaelisMenten equation, as described in Material and methods; So and S are expressed in gM and time (t) in h
Menten saturation kinetics (Fig. 2 B) with a K mof 45 gM (Fig. 2C), indicating that NO 3 uptake by sunflower plants is a carrier-mediated process. The depletion of NO z from uptake solution by sunflower plants is shown in Fig. 3 A. Uptake also exhibited saturation kinetics (Fig. 3 B). When the data of the NO~-depletion curve were analyzed by the integrated Michaelis-Menten equation, a Km value for NO~ of 23 ~tM was calculated (Fig. 3 C). Effect on anaerobiosis on the uptake and reduction of NO~ and NO2. External solutions were always bubbled
152
E. Agfiera et al. : Nitrate and nitrite uptake by sunflower
0.20
lOO 1.2 80 60
'-' o
40
b~
20
'-
o ~
0.15
rv
~.
u- O.8
>,'T : o.1o
g -6
o
b" ~o.~
ew
E
::1.
0.05
Z
0
A
i
4
6
Time
(h)
;
1
10
/+
5
;
7 pH
8
;
10
LL
"7
1.2
o E
~_
/\
B
"7 a~
1.2 0.8
o
i.J., Q/e
\
~0.8
P
o
7~b~2~ O"I
b~ ~0.~, Z
i60
\./, \ \
40
o -~-
u_ =m
g 20
/
~
O ~ O
20 '
t+O '
6"0
NO~ concenfrafion
80 ' 0
[wM}
9
/+
5
6
7
i
8
9
10
pH
Fig. 4A, B. Influence of pH on NO~ uptake and reduction (A) and on NO 2 uptake and reduction (B) in sunflower plants. Rates of NO~- and NO 2 uptake were measured in media buffered with 5mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes)-5 mM Mes adjusted at the indicated pH values. The NR and NiR activities in roots were determined in assay media buffered with 75 mM citrate-3-(N-morpholino)propanesulfonic acid (Mops)-glyeine buffer at the indicated pH values
o
i
." 2
/ -20
0
' 20
' 40
6'0
' 80
' 100
So-S / tn {S0/Sl Fig, 3. A Time course of NO 2- depletion of external solution by intact sunflower plants. B Plot of the rates of NO~- uptake versus NO~- concentration in the medium. C Hanes-Woolf representation of the integrated form of Michaelis-Menten equation for NO~ uptake. Experimental procedures were as in Fig. 2, except that NO 2 uptake was assayed
2. Effect of anaerobiosis on the uptake and reduction of NO~- and NO 2 by intact sunflower plants. Uptake solutions were bubbled either with air or with oxygen-free nitrogen gas. After a 6-h uptake period, NR and NiR activities were determined in the roots Table
Treatment
NO~- uptake experiment NO~ uptake rate
NO~ uptake experiment NR activity
NO 2 uptake rate
NiR activity
0.96 0.16
26.8 29.8
with air in order to maintain an adequate oxygen tension near the roots. When the solutions were made nearly anaerobic by continuous bubbling with N 2 instead of air, N O ~ uptake was fully abolished while N O ~ uptake greatly diminished (Table 2). This observation indicates that both NO~-- and N O 2 - u p t a k e systems are highly dependent on a continuous supply of metabolic energy. The removal of oxygen during the 6-h uptake period assayed did not substantially affect N R and N i R activities in the roots (Table 2).
Effect of p H on the uptake and reduction of NO 3 and NO2. Uptake and reduction processes of N O 3 and N O 2 were differently influenced by p H (Fig. 4). Uptake of both anions was higher at a mildly acid pH, maximal rates occurring at an external p H value around 5.5. In contrast, reduction of N O ~ and N O ] exhibited optimal pHs of 7.5 and 8.0, respectively.
g m o l - h - 1.g -1 F W Aerobiosis Anaerobiosis
1.24 ND a
" N D = n o t detected
0.20 0.17
Discussion
The development of accelerated N O S uptake following initial exposure to N O ~ has been observed in some
E. Agiiera et al. : Nitrate and nitrite uptake by sunflower plants (Tompkins et al. 1978; Deane-Drummond 1984; MacKown and McClure 1988). Nitrate uptake into nitrogen-starved sunflower plants also showed a lag phase (Fig. 1 A). Nevertheless, uptake was high and linear from the beginning when the plants were pretreated with NO 3 (Fig. 1 B). On the other hand, the presence of CHI during NO;- pretreatment prevented NO 3 uptake (Table 1), indicating that NO 3 induces de-novo synthesis of the NO;--uptake system, as previously found in other plants (Tompkins et al. 1978; Deane-Drummond 1984; Morgan etal. 1985b; MacKown and McClure 1988). We did not detect a low-capacity NO~-uptake system in nitrogen-starved sunflower plants comparable to that found in barley roots (Behi et al. 1988). The kinetics of NO 3 uptake by sunflower plants (Fig. 2) is consistent with a carrier-mediated mechanism with negligible passive uptake. This is corroborated by the complete suppression of NO 3 uptake when the plants were pretreated with CHI or NEM (Table 1). For the NO 3 concentration range used in this work (up to 0.2 mM), only a single uptake system with a Km of 45 gM was detected (Fig. 2). This value is within the range of Km values found in other higher plants either for single uptake system or for the mechanism I (high affinity) of dual systems (Beevers and Hageman 1983; Goyal and Huffaker 1986). In those uptake systems where both carriermediated and free-diffusion components were involved, CHI and sulfhydryl-group reagents abolished the former without affecting the latter (Serra et al. 1978; Kochian and Lucas 1982; Ibarlucea et al. 1983). The NO 3-uptake system of sunflower plants exhibited a lower sensitivity to pHMB than to NEM (Table 1). A similar response has been found for the NO~--uptake system of Chlamydomonas reinhardtii (C6rdoba et al. 1986) and sunflower (this paper). N-ethylmaleimide is known to permeate membranes more readily than pHMB (Kochian and Lucas 1982; C6rdoba et al. 1986). This agrees with the observation (Table 1) that NR, which contains SH-groups that are essential for its catalytic activity (Guerrero et al. 1981; Beevers and Hageman 1983), was inactivated in vivo in a greater extent by NEM than by pHMB. Sunflower plants were also able to develop a NOzuptake system when exposed to NO 3 or NO 2 (Fig. 1 B). Nitrite uptake was greatly prevented by CHI (Table 1), indicating that induction of the NO~-uptake system also involved de-novo protein synthesis. The existence of an inducible NO~-uptake system, scarcely studied in higher plants, has only been observed in wheat (Jackson et al. 1974; Goyal and Huffaker 1986) and barley (Ibarlucea et al. 1983). Nitrite uptake by intact sunflower plants exhibited Michaelis-Menten kinetics (Fig. 3), indicating a carrier-mediated mechanism. It was more susceptible to inactivation by NEM than by pHMB, as found for NO 3 uptake (Table 1) and also for the NOz-uptake system of the green alga Chlamydomonas reinhardtii (C6rdoba et al. 1986). Nevertheless, in contrast to NR, NiR from sunflower roots was not inactivated in vivo by NEM (Table 1), which agrees with the lower sensitivity of NiR to sullhydryl-group reagents found in vitro (Beevers and Hageman 1983). In contrast to NO;- uptake, NO~- uptake was not fully suppressed by pretreat-
153 ments with either CHI or NEM (Table 1), or when the uptake solution was made anaerobic (Table 2). This might indicate that some NO2 could enter into the root cells by a passive non-mediated mechanism. The observation that NO~ and NO~ uptake was drastically abolished when oxygen was removed from the uptake solution (Table 2) indicates that uptake is highly dependent on metabolic energy derived from root respiration. Ullrich (1987) has postulated that NO 3 is taken up by a 2H+/NO;- symport coupled to an ATPdependent proton-extrusion pump which generates the proton electrochemical gradient required for NO~ transport against a concentration gradient. We found that not only NO 3 uptake but also NO 2 uptake proceeded faster at acid pH, drastically decreasing at external pH values above 5.5 (Fig. 4A). According to the proposed 2H+/NO3 symport machanism (Ullrich 1987), which might be extensible to NO2 uptake, a high proton concentration in the external medium would build up the proton electrochemical gradient across the plasma membrane and, therefore, the driving force both for NO3 and NO~- uptake. Reduction of NO 3 and NO 2 exhibited different responses to pH, showing optimum pH values of 7.5 and 8.0, respectively (Fig. 4B). The results presented in this paper also show that in sunflower plants uptake and reduction processes, both of NO 3 and NO 2, exhibit different characteristics. First, the observed Km values for uptake are lower than the reported Km values for reduction (Guerrero et al. 1981 ; Beevers and Hageman 1983). Second, uptake and reduction proceed at different rates. Nitrate-uptake rates are four to six times higher than NO3-reduction rates in the roots. This indicates that a portion of NO 3 absorbed by the plant is accumulated in the root and- or translocated to the shoot. In corn roots taking up 15NOj after induction of NO 3-uptake system and NR activity, about 30% of the incoming 15NO 3 was reduced, 20% accumulated and 50% translocated (Morgan et al. 1985a). In contrast to NO 3 uptake, NOz-uptake rates are about 30 times lower than NO~-reduction rates in the roots, indicating that all the NO~ taken up by sunflower plants is reduced in the roots. In fact, NO2 was never detected in the root tissue. Third, anaerobic conditions greatly suppress NO;- and NO 2 uptake without substantially affecting their reduction. And fourth, uptake and reduction processes show different pH optima. This research was supported by grant PB86-0232 from the Direcci6n General de Investigaci6nCientificay T6cnica (Spain). One of us (E.A.) thanks the Consejeria de Educaci6n y Ciencia de la Junta de Andalucia for the tenure of a fellowship.We thank Miss G. Alcalfi and Miss C. Santos for their valuable technical and secretarial assistance. References Agiiera, E., de la Haba, P., Maldonado, J.M. (1987a) Induction of nitrate reductase, nitrite reductase and glutaminesynthetase by nitrate and/or nitrite in germinating sunflower cotyledons. Effect of plant hormones. Plant Physiol. (Life Sci. Adv.) 6, 255-258 Agfiera, E., de la Haba, P., Maldonado, J.M. (1987b) In vitro stabilization and tissue distributionof nitrogen-assimilatingenzymes in sunflower.J. Plant Physiol. 128, 443-449
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