P l a n t a 9 Springer-Verlag1990
The effect of endogenous and externally supplied nitrate on nitrate uptake and reduction in sugarbeet seedlings Gisela M~ick and Rudolf Tischner Pflanzenphysiologisches Institut der Universit/it, Untere Karspfile 2, D-3400 G6ttingen, Federal Republic of Germany Received 18 August 1989; accepted 2 April 1990
Abstract. The pericarp of the dormant sugarbeet fruit acts as a storage reservoir for nitrate, ammonium and ~-amino-N. These N-reserves enable an autonomous development of the seedling for 8-10 d after imbibition. The nitrate content of the seed (1% of the whole fruit) probably induces nitrate-reductase activity in the embryo enclosed in the pericarp. Nitrate that leaks out of the pericarp is reabsorbed by the emerging radicle. Seedlings germinated from seeds (pericarp was removed) without external N-supply are able to take up nitrate immediately upon exposure via a low-capacity uptake s y s t e m (Vma x = 0.8 gmol NO~-" (g root FW) - 1. h - 1 ; Ks = 0.12 mM). We assume that this uptake system is induced by the seed nitrate (10 nmol/seed) during germination. Induction of a high-capacity nitrate-uptake system (Vmax= 3.4 lamol NO~-'(g root F W ) - X.h- 1; K s = 0.08 mM) by externally supplied nitrate occurs after a 20-min lag and requires protein synthesis. Seedlings germinated from whole fruits absorb nitrate via a highcapacity uptake mechanism induced by the pericarp nitrate (748 nmol/pericarp) during germination. The uptake rates o f the high-capacity system depend only on the actual nitrate concentration of the uptake medium and not on prior nitrate pretreatments. Nitrate deprivation results in a decline of the nitrate-uptake capacity (tl/2 of Vmax= 5 d) probably caused by the decay of carrier molecules. Small differences in Ks but significant differences in Vmaxindicate that the low- and high-capacity nitrate-uptake systems differ only in the number of identical carrier molecules. Key words: Beta (nitrate reduction) - Fruit (N reservoir) - Nitrate reductase activity - Nitrate uptake - Pericarp (N reservoir) - Seed (nitrate uptake)
transport protein has not yet been identified; it is, however, assumed to operate in the plasmalemma (Ward et al. 1988). The nitrate-uptake system is substrate-inducible (Lee and Drew 1986) and the induction involves de-novo protein synthesis of the carriers (Goyal and Huffaker 1986; Jackson et al. 1973). The induced system is characterized by a high nitrate-uptake capacity (Clarkson 1986; Behl et al. 1988). Whether this high-capacity nitrate-uptake system is different from the low-capacity uptake system is not yet known. The low-capacity system is reported to be a noninducible, constitutive nitrate-uptake system as inferred from low uptake rates measured in plants that have never been exposed to nitrate (for review, see Clarkson 1986). Nitrate-free growth conditions, however, are hard to achieve. One has to be aware of a possible contamination of the chemicals used for nutrient solutions with trace amounts of nitrate (Heath-Paglusio et al. 1984). Solid growth substrates like sand or vermiculite might also contain nitrate (Funkhouser and Garey 1981). Besides this external nitrate, the endogenous nitrate of the fruits or seeds must be considered a possible inducer of nitrate uptake and reduction during germination. Sugarbeet fruits contain a rather high a m o u n t o f nitrate (Rehm 1953). We found 99% of the fruit nitrate in the dry pericarp and 1% in the dormant seed. To study the effect o f this pericarp nitrate in comparison with externally supplied nitrate on nitrate uptake and reduction we used sugarbeet seedlings that were either germinated from whole fruits or from seeds isolated from the pericarp.
Material and methods Plant material, germination, and hydroponic cultivation. Monogermic fruits of sugarbeet (Beta vulgaris L.), no. 3AO 166, were obtained
Introduction It is now well established that nitrate is absorbed via carrier proteins (Goyal and Huffaker 1986). The nitrateAbbreviations: NR = nitrate reductase; pFPA =para-fluorophenyl-
from Kleinwanzlebener Saatzucht (KWS) AG, Einbeck, FRG. Either the whole fruits (pericarp enclosing the seed) or the seeds isolated from the pericarp were germinated without external Nsupply on filter paper moistened with 0.5 mM CaSO4 at room temperature. The one-week-old seedlings were either directly used for the uptake experiments or further cultivated hydroponically in a modified Long Ashton solution (Mack 1988) with or without
G. M/ick and R. Tischner: Nitrate uptake and reduction in sugarbeet
nitrate, depending on the experimental conditions. Growth conditions were 20~ C/17~ (day/night) and 14h of light daily (180 Ixmol-m-2- s- 1).
Table 2. Nitrate-reductase activity (Ixmol NO~ .g FW-1 .h-l) of
the isolated sugarbeet seed after a 48-h imbibition of the whole fruit with or without nitrate In-vivo test
Net nitrate uptake Low-capacity uptake system. The basic uptake rates were measured with seedlings germinated from isolated seeds without external Nsupply. Prior to the uptake experiment the seedlings were transferred to the uptake solution for 4 min in order to saturate the free space of the root cell wall. Then the uptake experiment was started: the plants were exposed to the fresh, aerated uptake solution for 8 min; the nitrate concentration was as indicated (Fig. 1). Net uptake rates were calculated from nitrate depletion in the uptake medium.
High-capacity uptake system and nitrate pretreatment. To induce the nitrate-uptake system with externally supplied nitrate, seedlings germinated from isolated seeds were grown hydroponically for 3 d in a modified Long Ashton solution containing from 0.05 to 1.0 mM nitrate. The net uptake rates were measured in uptake solutions identical to the growth medium or with different nitrate concentrations. In the latter case, cell-wall saturation was achieved as mentioned above.
Nitrate determination. Nitrate in the uptake solutions was determined according to Thayer and Huffaker (1980) by high-performance liquid chromatography and ultraviolet-detection (210 nm). The anion-exchange column was packed with Partisil-10 SAX (Whatman, Maidstone, Kent, UK). Nitrate-reductase (NR) activity was measured in vivo (modified after Jaworski 1971) in 0.1 M potassium-phosphate buffer, pH 7.4, with 0.05% (v/v) Triton X-100. Unless stated otherwise, no nitrate was added to the incubation medium.
Distribution pattern of N-compounds in pericarp and seed. Both the dry pericarp and the d o r m a n t seed of the sugarbeet fruit contained inorganic and organic N-compounds (Table 1). The soluble protein content was about the same both in pericarp and seed on an organ basis, whereas the other N - c o m p o u n d s measured were significantly higher in the pericarp: nitrate was located almost exclusively in the pericarp; the a m m o n i u m and s-amino N contents were six- and fourfold higher in the pericarp than in the seed.
Effect of endogenous nitrate on NR activity of the germinating seed. Activity o f N R was detectable in the seed
Table 1. Distribution of N-compounds in pericarp and seed of the dormant sugarbeet fruit
Nitrate (nmol/organ) Ammonium (nmol/organ) Protein (~tg/organ) ~t-amino-N (~tg/organ)
7 mM nitrate
48 h after imbibition of the whole fruit; no significant difference in N R activity (measured in vivo without external nitrate supply) of the isolated seed was found after soaking the whole fruit with distilled water or with 7 m M nitrate (Table 2). Thus, soaking the fruit in externally supplied nitrate, in addition to the pericarp nitrate, did not increase seed N R activity. Two possible reasons for this result must be considered: (i) the pericarp supplies enough nitrate to the enclosed seed for full induction of N R activity or (ii) the externally supplied nitrate cannot penetrate through the pericarp into the enclosed seed. However, addition of nitrate to the in-vivo test medium resulted in a two-fold increase in N R activity of the isolated seed (Table 2). This points to a nitrate limitation of seed N R as long as the seed was entirely enclosed in the pericarp. Thus, pericarp nitrate was not accessible to the germinating seed unless the radicle had penetrated the pericarp and reabsorbed the nitrate leaking from the pericarp: 86% of the pericarp nitrate and 82% of the pericarp a m m o n i u m leaked into the medium during a 3-d imbibition of the fruit.
Nitrate uptake by the seedlings grown without pericarp.
Without nitrate With 25 mM nitrate
748 (99%) 978 (85%) 166 (51%) 2.40 (81%)
10 (1%) 167 (15%) 158 (49%) 0.55 (19%)
Seedlings germinated from isolated seeds without an external N-supply absorbed nitrate at a low rate (basic rate) immediately upon exposure to nitrate. This basic rate depended on the ambient nitrate concentration (Fig. 1) and was measurable only in the low ( < 1 mM) nitrate concentration range. The basic uptake system (low-capacity uptake system) was characterized by a Ks of 120/aM and a Vmax of 0.80 /amo1 N O 3 - (g root F W ) - 2. h-1 Long-term exposure (80 min) of these plants to nitrate resulted in increased uptake rates after a 20-min lag. This nitrate-caused induction was sensitive to parafluorophenylalanine (pFPA), but the basic uptake rate was not affected (Fig. 2). The induced uptake system (high-capacity uptake system) was characterized by a Ks of 80/aM and a Vmax of 3.42 /amo1 N O 3 . ( g root F W ) - 2 . h-1. The Vmax of the high-capacity uptake system was 4-times higher than that o f the low-capacity uptake system, while the affinity for nitrate was similar in both cases. Nitrate uptake via the high-capacity uptake system at nitrate concentrations higher than 1 m M (Fig. 3) was detectable only after a nitrate pretreatment (either pericarp nitrate or high ( > 1 mM) concentrations of externally supplied nitrate) for 8-10 d. With non-treated plants the uptake rates declined with increasing ( > 1 mM) external nitrate concentrations (data not shown).
G. Mdck and R. Tischner: Nitrate uptake and reduction in sugarbeet
o 1 z I
NO~ concentration of uptake solution (raM)
"S 0.4. o
0 - - 0
- - o - - O - - e - - o ~
0.2 o z;
min Fig. 2. Induction of the high-capacity nitrate-uptake system with externally supplied nitrate: time-course of the net uptake rates of sugarbeet seedlings grown from isolated seeds after exposure to a 80-pM nitrate solution with (o) and without (e) 1 mM pFPA
7,= 7 3 2 Z
7 a~ 4
Fig. 4. Turnover of the high-capacity nitrate-uptake system of sugarbeet seedlings: time course of the nitrate-uptake capacity (Vm~x) and affinity (Ks) dependent on the nitrate-deprivation period. The uptake system was induced by the pericarp nitrate
Nitrate uptake by the seedlings grown with pericarp. Seedlings raised from whole fruits without external nitrate supply absorbed nitrate immediately after transfer to nitrate-uptake solutions at rates similar to those o f nitrateinduced plants (Fig. 3). They absorbed nitrate from an external nitrate concentration range o f up to 5 mM. Exposure of the plants to different nitrate concentrations for 60 min did not alter the uptake rates (data not shown), indicating that a high-capacity uptake system was already fully induced by the pericarp nitrate.
uptake rates of nitrate-induced plants depended only on the nitrate concentration of the uptake solution and not on prior nitrate pretreatments: similar uptake rates were measured at a certain external nitrate concentration with plants pretreated with 50 pM nitrate, with 250 ~tM nitrate or with the nitrate concentration o f the uptake solution. The uptake rate was adjusted to the supplied nitrate concentration within a few minutes (time resolution was restricted to technical procedures of root rinsing and plant transfer) and was independent o f the presence of pFPA.
Turnover of the high-capacity uptake system. Transfer o f nitrate-induced plants into an N-free medium caused a decline in the nitrate-uptake capacity (tl/2 of Vmax= 5 d) without a change in nitrate affinity (Fig. 4). T1/z was independent of the pretreatment nitrate concentrations (0.05, 0.1, 1.0, 5.0 mM) and also of the pretreatment period (3-14 d; data not shown)9
Effect of pretreatments on the uptake rates. The nitrate-
days in N-free growth m e d i u m
Fig. 1. Low-capacity nitrate-uptake system (basic uptake system) of sugarbeet seedlings: net nitrate-uptake rates of seedlings grown from isolated seeds without external N-supply. Prior to the uptake experiment the root cell wall was saturated with nitrate for 4 min, and uptake rates were measured during the following 8 min of exposure of the plants to nitrate. For each nitrate concentration a new set of N-free-grown plants was used. v~,~=0.80 pmol.(g root FW) -~ .h-l; K~=0.12 mM
concentration of uptake solution ( m M )
Fig. 3. High-capacity nitrate-uptake system induced by pericarp nitrate: net nitrate-uptake rates of sugarbeet seedlings germinated from whole fruits for 8-10 d. After a 4-min exchange phase of nitrate in the root cell wall the uptake rates were measured during the 8 min following transfer of the plants to the uptake solution. For each nitrate concentration a new set of fruit-grown plants was used. Vmax= 4.18 pmol" (g root FW) - 1. h- t ; Ks = 0.11 mM
Effect of nitrate concentration on induction of the nitrateuptake system after N-starvation. A 5-d nitrogen starvation of seedlings grown from whole fruits resulted in a 60% decrease of the nitrate-uptake rates (Fig. 5). Transfer of the N-starved plants to nitrate solutions in the low concentration range ( < 1 mM) resulted in an increase of the uptake rates after a 20 min lag (Fig. 5); after 40-50 rain, constant rates were reached responding to the ambient nitrate concentrations. I f the N-starved
G. M/ick and R. Tischner: Nitrate uptake and reduction in sugarbeet
/- .o- ~+ _./"-+I
Fig. 5. Induction of the high-capacity nitrate-uptake system of su-
garbeet seedlings with low nitrate concentrations (+, 80 laM; o, 150 gM; o, 400 gM; ,t, 80 gM plus 1 mM pFPA) after 5 d of N-starvation. Seedlings grown from whole fruits were N-starved in an N-free growth medium
e ___~--- e------e
"8 .o v
z "8 ::&
h 6. Induction of the high-capacity nitrate-uptake system with 2 mM nitrate after 5 d of N-starvation. The uptake solution either contained (A) or did not contain (o) 20 gg-ml-1 cycloheximide. Seedlings grown from whole fruits were N-starved in an N-free growth medium Fig.
seedlings were exposed to 2 mM nitrate a lag of 2 h prior to induction was observed; constant uptake rates were established at about 5-6 h after transfer (Fig. 6). Discussion
The pericarp of the sugarbeet fruit contains germination inhibitors (Rehm 1953) which impose a stabilized, coatinduced dormancy (Bewley and Black 1985) on the seed. Besides this known function of the pericarp we found that it also acts as a storage reservoir for nitrate, ammonium and e-amino-N (Table 1). The ions were released from the pericarp into the medium during imbibition of the fruit and were reabsorbed by the emerging radicle. The constituents of the pericarp created a microenvironment adapted to the seedling's demands enabling an autonomous growth for 8-10 d after fruit imbibition. The N reserves of the seed (Table 1) were not sufficient for normal growth (M/ick 1988). The fact that the pericarp of the sugarbeet fruit (and to a small percentage also the seed) contains nitrate rais-
es the question as to whether nitrate uptake and reduction measured in seedlings that are germinated and grown without an external nitrate supply can be termed constitutive. A constitutive nitrate-uptake mechanism has been reported by several authors for different plant species (for review, see Clarkson 1986). The possibility of an induction by endogenous nitrate, however, was not taken into account. It is known that not only sugarbeet but also Picea (Peuke 1987) and Pennisetum (Ngambi et al. 1981) seeds contain nitrate. In order to employ germination conditions as nitratefree as possible we removed the pericarp from the fruit and germinated the isolated seeds on a filter paper moistened with 0.5 mM CaSO4. Seedlings grown from these seeds were able to absorb nitrate at a low rate immediately upon exposure (Fig. 1). We call this low-capacity uptake mechanism "basic system" instead of "constitutive" because we assume that this basic system was induced by the endogenous seed nitrate during germination. Exposure of seed-grown seedlings to nitrate solutions resulted in an increase of Vm,xafter a 20-min lag (Fig. 2). This induction of the high-capacity uptake system could be prevented by pFPA, indicating the requirement for de-novo protein synthesis (Cowie et al. 1959). Transfer of nitrate-induced plants (with the high-capacity uptake system) to an N-free solution resulted in a decline of Vmax (Fig. 4); Vma~approached zero, indicating that the low-uptake system was not maintained in an active state during N-starvation as assumed by Clarkson (1986). In both cases (nitrate-induction and N-starvation) the Ks remained unchanged. Therefore we assume that the Vma~of nitrate uptake is regulated via synthesis and decay of carrier molecules and that the low- and highcapacity uptake systems differ only in the number of active carriers. Behl et al. (1988) suggested that the low-capacity nitrate-uptake system provides a sensing system for nitrate availability in the soil and that the assembly of the highcapacity system is prompted only after the low-capacity system has imported nitrate. The pericarp nitrate of seedlings germinated from whole fruits for 8-10 d induced a high-capacity uptake system (Fig. 3). McKown et al. (1982) reported only low nitrate-uptake rates for maize roots with the endosperm still attached and increasing rates after exposure to nitrate. The high-capacity system in sugarbeet was, however, completely induced by the pericarp nitrate and exposure to externally supplied nitrate did not alter the uptake rates. Once established, the uptake rates of the high-capacity uptake system did not depend on the nitrate pretreatment but only on the actual nitrate concentration of the uptake medium (< 1 mM). No protein synthesis was required for the adjustment of the uptake rates to different nitrate concentrations, indicating an enzyme-like behaviour of the nitrate-uptake mechanism. Nitrate uptake from nitrate concentrations higher than 1 mM required a nitrate treatment period of 810 d. If the treatment period was shorter, the uptake rates declined with increasing nitrate concentrations (> 1 mM), probably as the result of competition of nitrate
G. Mack and R. Tischner: Nitrate uptake and reduction in sugarbeet for the c a r r i e r s (Tischner a n d L o r e n z e n 1979). We ass u m e t h a t a d d i t i o n a l carriers were s y n t h e s i z e d d u r i n g the n i t r a t e t r e a t m e n t p e r i o d . T h e d e m a n d for a d d i t i o n a l carriers is i n d i c a t e d b y a 2-h lag for the i n d u c t i o n o f the n i t r a t e - u p t a k e s y s t e m w i t h 2 m M n i t r a t e (Fig. 6) c o m p a r e d w i t h a 20-rain lag f o r the i n d u c t i o n w i t h nit r a t e c o n c e n t r a t i o n s b e l o w 1 m M (Fig. 5). This work was supported by a grant from Bundesministerium f/Jr Forschung und Technologie and by Kleinwanzlebener Saatzucht AG, Einbeck.
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