Journal of Experimental Botany, Vol. 54, No. 381, pp. 431-444,
January 2, 2003
© 2003 Oxford University Press
Diurnal variation in uptake and xylem contents of inorganic and assimilated N under continuous and interrupted N supply to Phleum pratense and Festuca pratensis
Received 8 May 2002; Accepted 19 September 2002
1 Institute of Grassland and Environmental Research, Aberystwyth Research Centre, Aberystwyth SY23 3EB, UK
2 Kvithamar Research Centre, N-7500 Stjørdal, Norway
3 To whom correspondence should be addressed. Fax: +44 (0)1970 828357. E-mail: james.macduff{at}bbsrc.ac.uk
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Compensation by dark-period uptake of NH4+ and NO3– in the grasses Phleum pratense L. and Festuca pratensis Huds. following N deprivation during the preceding light period was investigated in flowing solution culture under an artificial 10/14 h light/dark cycle. N was supplied as either NO3–, NH4+ or NH4NO3 at 20±5 mmol m–3, available continuously or only during the dark period, for 5–10 d. Intermittent N supply did not affect total daily N uptake, growth rate or net partitioning of dry matter. Net uptake and influx of NO3– varied similarly throughout the diurnal cycle when NO3– was supplied continuously, with a marginal contribution by NO3– efflux. Influx was significantly higher and efflux slightly higher following interruption of NO3– supply during the light period. Nitrate accounted for 80% of N in xylem exudate except between hours 6–9 of the light period when the amino acid concentration increased 3-fold, primarily as glutamine. Diurnal variation in relative NO3– uptake exhibited five phases of constant acceleration/deceleration, described reasonably well assuming NO3– influx was subject to metabolic co-regulation by NO3– and amino acid levels in the cytoplasmic compartment of the roots. Accordingly, influx is determined by variation in root NO3– levels throughout the dark period and the first half of the light period, but is down-regulated by increased amino acid levels during the second half of the light period. The sharp light/dark transitions affect transpiration rate and hence xylem N flux which, in turn, affect NO3– levels in the cytoplasmic compartment of the roots and the rate of NO3– assimilation in the shoot.
Key words: Amino acids, ammonium, diurnal variation, efflux, Gramineae, influx, ion uptake, nitrate, potassium, xylem composition.
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Introduction |
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N uptake by plant root systems under non-limiting N supply is determined by plant growth rate and the attendant ‘demand for N’ associated primarily with protein synthesis in dividing and expanding cells of leaves (Gastal and Nelson, 1994). The coupling between uptake and growth is most apparent when measured over time-steps of days or weeks and has been demonstrated convincingly for the uptake of NO3– (Touraine et al., 1994). Mediation between rates of uptake and growth processes is attributed to the state of N metabolite/substrate pools within the plant, with phloem-borne amino acids regarded as prime candidates for effecting metabolite regulation of uptake systems in the roots (Cooper and Clarkson, 1989; Clarkson, 1998), although their role has been questioned recently (Forde, 2002). Short-term variation in uptake rates will arise from perturbation of supply and demand side components, including external nutrient availability and environmental variables (e.g. temperature and light) affecting biosynthetic or transport processes within the plant via the energy and carbon substrate status. Many species have a considerable capacity to buffer against some of these perturbations, notably in nutrient supply, via a number of mechanisms including the mobilization of stored N acquired during periods of excess supply, and a facility for luxury uptake. For example, dry matter production by Lolium sp. is unaffected for at least 10 d following the termination of N supply (Clement et al., 1979; Jarvis and Macduff, 1989).
Significant diurnal fluctuations in net uptake of N occur under most environmental conditions, but how these are coupled to, or regulated by, variation in N demand or C supply remains incompletely described (Matt et al., 2001; Glass et al., 2002). Fluctuations in NO3– uptake have been described more frequently (Pearson and Steer, 1977; Clement et al., 1978b; Hansen, 1980; Steingrover et al., 1986; Le Bot and Kirkby, 1992; Delhon et al., 1995a; Matt et al., 2001) compared with NH4+ (Hatch et al., 1986; Gazzarini et al., 1999), or the concurrent variation in NH4+ and NO3– uptake, where there may be an element of co-regulation (Breteler, 1973; Macduff et al., 1997). The amplitude and form of these fluctuations depend on light-regime and also vary with species. Commonly, uptake increases until the mid-point of the light period and then decreases throughout the dark period.
The relative importance of variation in influx and efflux to diurnal fluctuation in net uptake is not well characterized. The dark-induced decline in net NO3– uptake by Glycine max L. Merr. is attributable to down-regulation of influx (Delhon et al., 1995a). Zea mays L. seedlings exhibit no significant variation in NO3– efflux or influx, whilst efflux by Pennisetum americanum L. seedlings increases during the dark period (Pearson et al., 1981). At the molecular level, recent studies have demonstrated a correlation between diurnal patterns of N uptake and transcript abundance for the high-affinity NRT2 and AMT1 genes (Glass et al., 2002).
It has been argued that uptake rates by many species are normally regulated at well below their maximum capacity when the N supply is adequate (Touraine et al., 1994). The occurrence of rapid transient increases in uptake rates when N is resupplied after a short period of deprivation (Jarvis and Macduff, 1989; Lee and Rudge, 1986) supports this view. However, compensation for temporal variation in nutrient availability has been studied relatively infrequently compared with compensation for spatial variation (Robinson and van Veuren, 1998). To some extent this reflects the difficulty of controlling fluctuations in nutrient availability and measuring uptake simultaneously, even in solution culture. Hence, almost all available information on N uptake following N starvation is restricted to a single cycle of N deprivation and resupply, and to the initial rate of uptake, rather than the degree of compensation achieved in the longer term. When Lolium perenne L. cv. S23 was supplied with 7 mmol m–3 NO3– intermittently (3 d on 3 d off) over 42 d (Clement et al., 1979) the final shoot dry weight was significantly lower compared with plants continuously supplied. Uptake of NO3– was always enhanced following resupply, but beyond the first 6 d cycle this was not sufficient to compensate completely for the 3 d interruption in N supply.
The aims of the present study were (a) to assess the extent of compensation in uptake of NH4+ and NO3– by two grasses, Phleum pratense L. and Festuca pratensis Huds. after terminating the supply for the duration of the light period, and (b) to investigate the diurnal regulation of uptake by examining the external and internal fluxes of inorganic and assimilated nitrogen during and following interruptions in supply. Diurnal variation in NO3– and NH4+ net uptake by these species under continuous N supply has been described previously (Macduff et al., 1997) and the current experiments were performed under comparable conditions in flowing nutrient solutions, with N supplied at 20 mmol m–3 as NO3–, NH4+ or NO3–+NH4+, either continuously or intermittently (only during the dark period of the daily cycle) over 5–10 d. The inclusion of a combined NH4++NO3– supply enabled the assessment of interactive effects on compensatory changes in total N uptake, as external NH4+ inhibits NO3– uptake by many species (Lee and Drew, 1989; Jackson and Volk, 1995), whilst NO3– can both inhibit or promote uptake of NH4+ (Clarkson et al., 1986, 1992; Saravitz et al., 1994). In the experiment with NH4+ as the sole form of N the uptake of K+ was also measured because K+ uptake is inhibited by NH4+ (Jackson and Volk, 1995), although NH4+ and K+ transport across the plasma membrane may share common ion channels (Lee and Ayling, 1993).
The technique for supplying N ensured that uptake was ‘demand-limited’ as opposed to ‘supply-limited’ (Ingestad and Ågren, 1992) and from micromolar concentrations associated primarily with the activity of the high-affinity transporter systems (Crawford, 1995). The net uptake rates were measured in situ, without physical disturbance of plants, thereby avoiding artefacts associated with ‘transfer-shock’ (Bloom and Sukrapanna, 1990; Delhon et al., 1995a) or variable pH (Moritsugu et al., 1983) which may occur when plants are transferred from one solution to another. However, the NO3– influx assay necessitated the transfer of plants, although these were acclimated to the continuous bulk flow of solution through the root systems in the system of flowing solution culture.
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Plant material and culture conditions
The same batch of seeds of timothy (Phleum pratense L. cv. Bodin) and meadow fescue (Festuca pratensis Huds. cv. Salten), both from Norway, were used in all experiments. In each case seeds were sown directly into culture units of a system of flowing solution culture in a greenhouse in Aberystwyth, UK, incorporating the automatic control of concentrations of NO3–, NH4+, K+, and H+ in solution (Clement et al., 1974; Hatch et al., 1986). Each culture unit held 200 dm3 of recirculating nutrient solution, and 24 culture vessels each containing 10–12 plants following thinning of seedlings on emergence. The initial composition of nutrient solutions was (mmol m–3): NO3–, 250; K+, 250; Ca2+, 344; SO42–, 424; Mg2+, 100; H2PO4–, 50; Fe2+, 5.4, with appropriate micronutrients (Clement et al., 1978a), and solution pH 6.0±0.5. The entire solution was replaced every 2 or 3 weeks during the pretreatment growth period. During the experimental treatments the nutrient concentrations were controlled automatically, with concentrations of NO3–, NH4+ and K+ measured in each culture unit every 13 min (Experiments 1 and 3) or 27 min (Experiment 2) and maintained at the desired set-point concentrations, respectively, by automatic additions of Ca(NO3)2, (NH4)2SO4 and K2SO4, with all other nutrients supplied in proportion to that of NO3– or NH4+. The solution pH of 6.0±0.1 was maintained by automatic titration of Ca(OH)2/H2SO4. The air temperature was 25/15 °C (±3 °C) day/night and solution temperature was 19–21 °C throughout.
Lighting regime
Seedlings were established under natural illumination until day 42 (Experiment 1), day 48 (Experiment 2) and day 22 (Experiment 3). Thereafter, supplementary light was introduced progressively during natural daylight hours, initially provided by a single 400 W SON-T lamp (Philips) and then additionally by a 400 W HPI-T (Philips) lamp, suspended 1.5 m above the surface of each culture unit. Five days prior to imposing the intermittent N supply treatments, a ‘step’ light/dark regime was imposed in each experiment, consisting of 10 h light (08.00–18.00 h) and 14 h dark period, with light provided by a 400 W HPI-T and 400 W SON-T (Philips) lamp suspended 1.5 m above the surface of each culture unit, producing 550± 50 µmol m–2 s–1 PAR at the top of the plant canopy. This gave a daily quantum input of 18 mol m–2 d–1, reasonably close to a generally quoted saturation value of 20 mol m–2 d–1 with respect to relative growth rate by herbaceous species (Poorter and Van der Werf, 1998). At the end of the ‘light’ period, each culture unit was covered by a light-exclusion box (1.1x1.2x0.8 m), which allowed ventilation but reduced light levels to <0.02 µmol m–2 s–1 PAR. Radiation was logged by a Kipp solarimeter on an hourly basis. The short photoperiod was chosen to ensure that generative development was not induced, and no flowering stems were visible in either species during the experiments.
Measurement of net uptake
Net uptake of NH4+, NO3– and K+ were measured in situ on an hourly basis by the amounts of ions automatically delivered to culture units each hour to maintain set-point concentrations of each ion at 20± 5 mmol m–3 in solution. Rates were expressed per plant or on a unit root fresh weight basis and, where appropriate, the primary data sets were smoothed by a Savitzky–Golay procedure (Fig. P, Biosoft, using a single iteration with cubic polynomials fitted to successive five-point ‘windows’). In some instances data are presented as mean rates over successive 3 h intervals.
Experiment 1: uptake from intermittent NH4++NO3– supply
Two culture units each of P. pratense and F. pratensis were grown from seed (sown 4 August) under the conditions described above, until day 53 after sowing when the N supply was changed to NH4++NO3–. Thereafter the concentrations of both these ions were independently controlled at 20±5 mmol m–3, and K+ controlled at 20±5 mmol m–3. On day 56 an intermittent N supply was imposed on one of each pair of culture units per species by terminating the supply of NH4++NO3– for the entire light period (08.00–18.00 h), and resupplying N at the start of the dark period (DN treatment). Plants in the other two culture units were supplied continuously with N (CN treatment). Treatments continued for 10 d, but from day 7 onwards the supply of NH4+ to all culture units was terminated permanently and N subsequently supplied solely as NO3–. Plants were harvested for growth analysis at intervals: four culture vessels (44 plants) per culture unit on days 0, 7 and 10 of treatments. Plant and tiller numbers per vessel were counted, shoots separated from roots, fresh weights recorded and plant fractions freeze-dried prior to reweighing. Dried fractions were ground and analysed for total N by continuous flow mass spectroscopy (Twenty-twenty, Europa Scientific Ltd, Crewe, UK) linked to a C/N analyser (Roboprep CN, Europa Scientific Ltd, Crewe, UK).
Experiment 2: uptake from intermittent NH4+ supply
Four culture units of P. pratense were grown from seed (sown 16 August) as described above until day 47 after sowing when the N supply was changed to 20±5 mmol m–3 NH4+, with K+ controlled at 20±5 mmol m–3. The plants were allowed to acclimate for 7 d. On day 54 an intermittent (dark-period only) NH4+ supply was imposed, similar to that in Experiment 1, on two replicate culture units (DAm treatment). The other two units received a continuous supply (CAm treatment). Treatments continued for 5 d. Plants were harvested at the start and end as described above for Experiment 1.
Experiment 3: uptake from intermittent NO3– supply
Eight culture units of P. pratense were grown from seed (sown 11 October) as described above until day 49 when the N supply was changed to 20±5 mmol m–3 NO3–, with K+ controlled at 20± 5 mmol m–3. The plants were acclimated over 4 d and on day 53 a regime of intermittent (dark-period only) NO3– supply was imposed on four culture units (DNi treatment), similar to that in Experiment 1. The other four units received a continuous N supply (CNi treatment). Treatments extended over 5 d and plants were harvested from two culture units of each treatment at the start and end as described for Experiment 1. Plants in the other four culture units were reserved for the N flux measurements described below.
Diurnal variation in N fluxes and xylem composition under intermittent NO3– supply
Diurnal variation in NO3– influx, efflux and xylem N composition was compared for P. pratense under DNi and CNi treatments in Experiment 3. Hourly measurements were made over 24 h from the start of the light period (08.00 h) on day 3 of the treatment period.
Nitrate influx was measured by a 15N pulse-labelling technique. Two culture vessels of plants (n=22 plants) from each treatment (one vessel removed per replicate pair of culture units) were removed from the treatment units at 1 h intervals, allowed to drain for 15 s and then placed in a culture unit containing 300 dm3 of flowing nutrient solution of similar composition but containing 20 mmol m–3 NO3– labelled at 99.7 atom% 15N. All other environmental conditions were identical. The plants were 15N-labelled for 10 min and then the vessels removed and 15N-label washed out of the root ‘free space’ by transferring for 30 s into an identical culture unit containing unlabelled 20 mmol m–3 NO3–. Vessels were drained for 60 s before separating roots from shoots, recording fresh weights and freeze-drying. After reweighing the dry fractions were ground and analysed for total N and 15N by continuous flow mass spectroscopy (Twenty-twenty, Europa Scientific Ltd, Crewe, UK) linked to a C/N analyser (Roboprep CN, Europa Scientific Ltd, Crewe, UK).
Influx and translocation of 15N were calculated and expressed on a root fresh weight basis (Bakken et al., 1997). Efflux of NO3– was calculated as the difference between the concurrently measured hourly influx and net uptake rates, the latter given by the mean rate measured automatically on two separate full replicate culture units of plants (n=528 plants) per treatment in Experiment 3.
Transpiration rate was measured each hour by removing two culture vessels per treatment to plastic beakers containing 0.5 dm3 of aerated nutrient solution of similar composition as the flowing solutions and measuring weight loss after 30 min. The vessels were replaced in their original culture units afterwards and never used twice, or for the measurement of influx or xylem exudate composition.
Xylem exudate was also freshly collected on an hourly basis. On each occasion plants (n=11) in one culture vessel per treatment were defoliated in situ by cutting horizontally with a scalpel at a shoot height of 4 cm above the shoot/root junction. The ‘cut ends’ of the remaining stubble (leaf sheaths, leaves and pseudostems) were immediately blotted dry with a paper tissue, and xylem exudate collected continuously by a pasteur pipette over 30 min. The plants were discarded afterwards and exudate samples weighed and stored at –80 °C prior to analysis. Subsamples of exudate were taken for the analysis by automated colorimetry of NO3– (Henriksen and Selmer-Olsen, 1970) and NH4+ using a modified Bertholet reaction (Verdouw et al., 1977). Free amino acids in ultrafiltrates deproteinized with 1.22 vol. of 470 mol m–3 sulphosalicylic acid were determinied on a Chromaspek M J185 MKI amino analyser (Hilger Analytical Ltd, Margate, Kent) using post-column reaction with ninhydrin, and a gradient elution system using two lithium buffers pH 1.9 and 11.5. Results were expressed as mmol m–3 N in exudate.
Statistical analyses
Data for plant dry weights, tiller numbers and cumulative net uptake of N and K over the treatment periods were subject to analysis of variance with species and timing of N supply as class variables. Mean values for NO3– influx and efflux in Experiment 3 were compared by two-tailed t-tests with common variance.
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Growth response to intermittent N supply
Restricting the supply of N to the dark period (DN treatment) did not significantly (P >0.05) affect total dry matter production or the partitioning of growth as indicated by shoot:root ratios, compared with plants under a continuous supply (CN treatment) in any of the experiments. However, the final dry weights of DN plants were, on average, lower than those of CN plants of both species after 10 d of treatment in Experiment 1 (Table 1). Shoot:root ratios were significantly higher in F. pratensis than P. pratense.
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Uptake from intermittent NH4++NO3– supply
In Experiment 1 both P. pratense and F. pratensis increased uptake of N during the dark period to compensate fully for the withdrawal of the N supply during the preceding light period. Total uptake of NH4++NO3– over the 10 d of treatment was not significantly different between DN and CN plants (Table 2). Hence, compensation in NH4+ and NO3– uptake by DN plants were of the same overall magnitude during the dark period. The rate of NH4+ uptake by DN plants of both species during the first 3 h of the dark period was much higher in both absolute and proportional terms compared with CN plants and also compared with the uptake of NO3– by DN plants (Fig. 1A, B). Subsequently, NH4+ uptake by DN plants was severely down-regulated and more so in P. pratense than F. pratensis, accounting for the lower total daily uptake of NH4+ by the former species. Consequently, maximum hourly rates of NH4+ uptake were far higher in DN than CN plants.
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The ratio of NH4+:NO3– uptake (Fig. 1C) was almost always >1.0, and was higher throughout the diurnal cycle in F. pratensis than P. pratense, suggesting significant genotypic variation in the extent of preferential uptake of NH4+. It also declined progressively in CN plants of both species, from a maximum at the start of the light period to a minimum 4 h into the dark period. DN plants had relatively high ratios at the start of the dark period compared with CN plants, indicating an increase in the degree of preferential uptake of NH4+, but thereafter the ratios were more or less similar in both treatments.
The degree of compensation in N uptake by DN plants achieved during the dark period is illustrated by the fact that CN plants absorbed approximately 40% of their total daily uptake of NO3– uptake and 50% of their NH4+ uptake during the 10 h light period (Table 3). Consequently, on a relative basis the compensation by DN plants was greater for NH4+ than NO3– uptake. Interestingly, P. pratense absorbed a significantly higher proportion of its total uptake of NH4+ during the light period compared with F. pratensis.
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Uptake from intermittent NH4+ supply
When NH4+ was the sole form of N supplied (Experiment 2) its uptake following resupply at the start of the dark period (Fig. 2A) showed a very similar pattern to that observed when NH4++NO3– were both supplied. The rate of NH4+ uptake was highest during the first hour, declining rapidly over the subsequent 2–4 h and then at a slower rate over 4–6 h. Summed over the treatment period the increase in uptake of NH4+ achieved by DAm plants under darkness fully compensated for withdrawal of the supply during the light period (Table 2).
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The total quantities of K+ absorbed over the 5 d of treatment were not significantly different (P >0.05) for CAm (236 µmol plant–1) and DAm (231 µmol plant–1) plants. However, there were significant differences (P <0.001) between the two treatments in the diurnal pattern of K+ uptake, with CAm plants absorbing a much higher proportion (58±4% compared with 22±2%) of their daily uptake of K+ during the dark period. Hence, withdrawal of the NH4+ supply during the light period caused a dramatic increase in rates of K+ uptake by DAm plants compared with CAm plants (Fig. 2B). The high rates of K+ uptake continued until NH4+ was resupplied and subsequently declined rapidly to zero or thereabouts within 2–3 h. Net efflux of K+ from DAm plants into the flowing nutrient solutions occurred frequently towards the end of the dark period.
Coincidental rates of NH4+and K+ uptake were poorly correlated both in CAm (r2=0.17) and DAm (r2=0.40) plants, although the rate of K+ uptake for a given rate of NH4+ uptake was generally higher for CAm than DAm plants.
Uptake from intermittent NO3– supply and diurnal fluctuations in NO3– influx and efflux
With NO3– as the sole form of available N (Experiment 3) uptake following its resupply at the start of the dark period fully compensated for its withdrawal during the light period (Table 2). Rates of NO3– uptake by DNi plants exhibited distinct oscillations during the dark period on several days (Fig. 3).
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Diurnal variation in nitrate influx and efflux by DNi and CNi plants were measured on day 3 in Experiment 3, with efflux estimated as the difference between the measured net uptake and influx. No estimates of efflux from DNi plants were made during the light period when the NO3– supply was terminated. However, ‘potential influx’ by these plants was measured by the 15N technique.
Under continuous NO3– supply (CNi plants) the diurnal pattern in influx was broadly similar to that for net uptake (Fig. 4A), the rate increasing during the first 6–8 h of the light period and then decreasing more or less progressively until mid-way through the dark period, after which point it remained stable. However, the amplitude of the diurnal fluctuation appeared to be greater for net uptake than influx, in so far as net uptake increased more acutely during the first half of the light period, and decreased more acutely during the first half of the dark period. Both influx and net uptake started to decline before the end of the light period, as opposed to during the dark period. Hence their decline was not induced per se by darkness.
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The ‘difference method’ used to estimate efflux resulted in several small negative values (i.e. where net uptake apparently exceeded influx), attributed to sampling errors. However, the general trend was evident and compared with influx, efflux was a relatively minor component of net uptake by CNi plants throughout the diurnal cycle (Fig. 4A), with means of 0.20 and 0.07 µmol N h–1 g–1 root f.wt, respectively, for light and dark periods (Table 4). On the basis of sequences of three or more hours during which influx exceeded net uptake, efflux was highest during the first half of the light period and between hours 5–9 of the dark period.
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Influx and ‘potential influx’ of NO3– was significantly higher in DNi plants than CNi plants throughout most of the diurnal cycle (Fig. 4B), generally by a wider margin during the dark period than the light period (Table 4). Although influx was of the same order as net uptake by DNi plants during darkness, the efflux component (mean=0.48 µmol N h–1 g–1 root f.wt) was slightly higher compared with CNi plants although the difference was not statistically significant (Table 4). It was also notable that during the first 4 h of the dark period, NO3– influx consistently exceeding net uptake by DNi plants, but vice versa in CNi plants.
Diurnal fluctuations in xylem N composition
The procedure for sampling xylem exudate resulted in a very few samples of insufficient volume for amino acid analysis. Throughout the diurnal cycle (Fig. 5) xylem N concentration declined in the order: [NO3–]x >[amino acid]x >[NH4+]x, with the exception of 2 h towards the end of the light period when concentrations of amino acid-N increased sharply (Fig. 5C).
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In plants supplied with NO3– only during the dark period (DNi) the diurnal variation in [NO3–]x and [NH4+]x exhibited three phases, and most distinctly in the case of NO3– (Fig. 5A, B). Phase 1 extended over the entire 10 h light period, during which [NO3–]x declined almost linearly to a minimum value for the diurnal cycle. Phase 2 covered hours 1–5 of the dark period, during which [NO3–]x. increased steeply and almost linearly. Phase 3, extending throughout the remainder of the dark period was characterized by relative stability in [NO3–]x although [NH4+]x increased progressively until the last hour of the dark period.
The diurnal pattern of [NO3–]x in plants continuously supplied with NO3– (CNi) differed in several respects from that in DNi plants. Firstly, the diurnal amplitude was smaller. Secondly, concentrations measured during the light period were higher than in the corresponding DNi plants and did not decline consistently over time. Thirdly, the increase in concentration following the onset of the dark period (Phase 2) was relatively modest and concentrations remained lower in CNi compared with DNi plants throughout the dark period.
By contrast with NO3–, the diurnal variation in total xylem amino acid concentrations, [amino acid]x, was very similar in both N treatments, with the exception of several hours towards the end of the dark period (Fig. 5C). This was also true for the specific amino acids in the exudate. Glutamine accounted for well over 50% of the total amino acid N, with asparagine and glutamic acid the only other species regularly exceeding 5% in terms of proportional contribution (data not presented). Averaged throughout the diurnal cycle the ten most abundant species in the exudate of CNi plants were, in decreasing order: Gln, Glu, Asn, Ser, Orn, Ala, Gly, Val, Arg, Lys. In Dni plants the order was: Gln, Asn, Glu, Ser, Ala, Lys, Arg, Orn, Val, Thr. In all cases the mean concentrations of individual amino acids over the 24 h period were not significantly different (P >0.05) in DNi and CNi plants.
The most remarkable feature of the diurnal variation in [amino acid]x was the 3-fold increase in amino acid concentrations between hours 6–8 of the light period, followed by an equivalent decline between hours 8–10. In absolute terms this transient increase was dominated by glutamine, which doubled in concentration, although there were several fold increases in a number of the less abundant species (i.e. Asp, Thr, Ser, Asn, Glu, Ala, Gaba, Lys, Arg).
The final 5 h of the dark period was the only part of the diurnal cycle during which DNi rather than CNi plants differed consistently in [amino acid]x with the higher values in DNi plants attributable almost entirely to glutamine concentrations which were twice as high in DNi plants. Interestingly, concentrations of glutamic acid were 50% lower in DNi than CNi during this period. Norleucine, a quantitatively minor component of the xylem exudate was the only amino acid present exclusively in the dark period, with the exception of one sample collected during the light period. It was consistently detected between 23.00–07.00 h in CNi plants and intermittently in DNi plants.
Diurnal fluctuations in xylem N flux
The proportion of the 15N-label absorbed during the 10 min 15NO3– influx measurements that was rapidly translocated to the shoot prior to harvesting the plants showed a distinct diurnal pattern (Fig. 6A), similar in CNi and DNi plants. The proportion was 2–3-fold higher during the light period and in the case of CNi plants it began to decline 2 h prior to the onset of the dark period. Given the marked decline in 15NO3– influx during darkness (Fig. 4A), the absolute decline in 15N translocated (data not presented) was far greater than the proportional decline during darkness. However, it should be stressed that this is the pattern for translocation of N immediately (<12 min) following its uptake by the roots, which constitutes only a fraction of the total N translocated in the xylem at any time.
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Transpiration loss expressed on a unit leaf f.wt basis (Fig. 6B) was similar for CNi and DNi plants (P >0.05) and varied very little during the light period before declining to a very low value following the onset of darkness. The rate increased gradually over the second part of the dark period.
Although absolute xylem fluxes of NO3– and amino acids were not measured the approximate trends over time can be inferred as the products of successive xylem exudate concentrations and transpiration rates. Because transpiration varied relatively little within either the light or dark periods, the error inherent to this approach is likely to be more or less constant during each of the two periods, hence variation in xylem exudate concentration within the light period reflects variation in xylem flux, and similarly within the dark period. Accordingly the large difference in transpiration between the light and dark periods implies that xylem N flux during the light period was several fold higher than during the dark period, despite the higher xylem N concentrations measured during the darkness. Support for this inference is also provided by the diurnal pattern of 15N translocation (Fig. 6A).
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Compensatory uptake of N under intermittent supply
Restricting the N supply to the dark period within the diurnal cycle appears to have no adverse effect on the growth of these two species of grass over the relatively short time-scale considered in these experiments. Irrespective of the form in which N was supplied, the increased N uptake during the dark period fully compensated for the withdrawal of the N supply during the preceding light period. These results are consistent with the view that under conditions of continuous adequate N supply the uptake systems in these species normally operate well below their maximum kinetically defined capacities (Touraine et al., 1994). Furthermore, they indicate that the decline in transpiration during darkness does not in itself play any role in a systemic or whole plant regulation of uptake. Rather, at this level uptake is determined by the internal demand for N and, when necessary, for example following a period of N deprivation, the ‘normal’ diurnal changes in uptake are modulated accordingly.
There is no evidence that compensatory increases in NO3– uptake exceed those for NH4+ uptake. In fact when supplied continuously with N, a lower proportion of the total daily uptake of NH4+ occurred during the dark period compared with NO3– uptake, suggesting both in absolute and proportional terms that the compensatory increase in NH4+ uptake during darkness was higher than for NO3–. The difference between the two ions appears to be particularly marked during the first 2–3 h of the dark period, with a much greater up-regulation of the NH4+ uptake system irrespective of the presence of NO3–. Hence, NH4+ uptake by these species was not inhibited by darkness per se.
The more acute increase in NH4+ uptake at the start of the dark period compared with NO3– uptake when these ions were resupplied together following deprivation, could be associated with the accumulation of carbohydrate in the roots during the preceding light period, facilitating rapid assimilation of newly absorbed NH4+. Alternatively, it might arise from initially low amino acid and NH4+ concentrations in the root cytoplasmic compartment enabling the rapid up-regulation of uptake. The differences in the initial uptake rates of NH4+ and NO3– following resupply also suggests some degree of differential regulation, perhaps via different effectors or sensitivities to a common effector. Studies of longer term interactions between NH4+ and NO3– uptake by L. perenne (Clarkson et al., 1986, 1992) suggest that NO3– uptake is inhibited in the presence of NH4+ whilst NH4+ uptake is stimulated by NO3–. Consequently, the high initial rate of NH4+ uptake by DN plants during the dark period could itself have delayed the up-regulation of NO3– uptake, via a transient increase in cytoplasmic concentrations of NH4+ or amino acid products of NH4+ assimilation, prior to their translocation to the shoots.
Interruption of N supply to L. perenne for 3 d out of every 6 d (Clement et al., 1979) resulted in a subsequent increase in NO3– uptake relative to plants receiving a continuous N supply, but failed to compensate fully for the interruption in N supply beyond the first 6 d cycle. The critical period of interruption beyond which full compensation in N uptake is not achieved presumably lies between 10 h and 3 d, subject to inter-specific variation.
Diurnal variation in NO3– influx and efflux
The results demonstrate that diurnal variation in net uptake of NO3– by P. pratense from constant low concentrations of NO3– is associated predominately with changes in influx rather than efflux. Likewise the compensatory increase in net uptake occurring after interruption in the NO3– supply during the light period is attributable mainly to the influx component. This is consistent with the view that influx is the most important site of regulation at external concentrations associated with the high-affinity NO3– transporter systems (Siddiqi et al., 1990; Lee, 1993; Touraine et al., 1994), although efflux has not been characterized sufficiently for its regulatory significance to be assessed categorically (Glass et al., 2002). Down-regulation of NO3– influx to match plant demand is regarded as advantageous in avoiding expenditure of energy to generate –µH+ across the plasma membrane (Lee, 1993). The decline in net NO3– uptake by Glycine max L. Merr. during the dark period is also almost entirely associated with down-regulation of influx (Delhon et al., 1995a, b), attributable either to accumulation of NO3– and asparagine in the roots, or to a decrease in shoot assimilation of NO3– leading to a decrease in phloem transport of carboxylates to the roots which, in turn, could affect the availability of HCO3– for an HCO3–/NO3– antiport (Ben Zioni et al., 1971). However, the subsequent increase in influx into Glycine max during the light period is accompanied by an increase in efflux.
The mean ratios of influx:efflux for CNi plants in the present study were, respectively, 24 and 53 during the light and dark periods, suggesting efflux is least significant during darkness. However, these ratios are several fold higher than those measured by 13N techniques for Lolium multiflorum Lam. at a single point within the diurnal cycle, (Macduff and Jackson, 1992). Furthermore, DNi plants had lower influx:efflux ratios than CNi plants during the dark period, consistent with the hypothesis that regulation of net uptake via efflux might be more important in the short-term and during recovery from nutrient stress (Deane-Drummond, 1990; Lee, 1993). Nevertheless, the relatively minor contribution of NO3– efflux to diurnal variation in net uptake by P. pratense is clearly at odds with the model of constant influx and variable efflux invoked to explain diurnal variation in NO3– uptake by L. perenne (Scaife, 1989) although this gave close agreement between predicted and actual variation in net uptake.
Recent evidence from molecular studies suggests that NO3– itself is responsible for inducing gene expression associated with iHATS, but that its downstream metabolites are responsible for regulation in terms of transcript abundance (Glass et al., 2002). The ‘potential influx’ data for DNi plants in the present study suggest that iHATS remained induced throughout the –N light period (i.e. influx still reasonably high). Hence it is unlikely that the subsequent increase in actual influx during the first 3 h of the dark period was an induction effect arising from the increase in cytoplasmic NO3–. It is also difficult to explain this increase in influx on the basis of post-transcriptional regulation by cytoplasmic NO3–, as advocated in the past by several authors (Glass et al., 2002), because the net NO3– content of the roots probably increased with increasing influx, given the high influx and low estimated xylem NO3– flux. However, it is conceivable that recently absorbed NO3– was transported rapidly out of the cytoplasmic compartment of the roots into the vacuolar compartment during this initial 2–3 h period, with the balance loaded into the xylem, thereby maintaining very low root cytoplasmic concentrations of NO3–. The occurrence of transiently high ‘vacuolar demand’ for N has been inferred from the unusually high rates of NO3– uptake by N-starved L. perenne subject to severe defoliation at the same time as NO3– is resupplied (Macduff et al., 1989). The concurrent translocation of 15N-absorbed during the NO3– influx assays indicates that significant translocation of recently absorbed NO3– continued for 1 or 2 h into the dark period. The decrease in NO3– influx by DNi-treated plants measured after 3 h of darkness could denote the slowing down or cessation of this vacuolar compartment filling phase in the roots, with down-regulation of influx attributable to a subsequent increase in NO3– levels in the cytoplasmic compartment.
Diurnal variation in vascular N composition and fluxes
Translocation of NO3– in the xylem and its reduction in leaves is believed to be more sensitive to light/dark transitions per se compared with NO3– uptake by the roots (Deane-Drummond, 1990; Delhon et al., 1995a, b). This view is supported by the present data for P. pratense, in so far as the transition from light to darkness (and by implication from darkness to light) precipitated sharper immediate (<1 h) changes in the estimated xylem flux of NO3– compared with uptake (Fig. 7). These rapid changes in xylem flux are attributable to the changes in transpiration. However, variation in transpiration was not a factor in determining the further substantial variation in NO3– uptake and xylem N fluxes occurring during the light or dark periods.
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Amino N cycling between shoots and roots via phloem and xylem may provide an integrating mechanism for the regulation of N uptake by plant demand for N (Cooper and Clarkson,1989). Under this scheme, as metabolic demand for assimilated N increases, the cycling flux of amino acids will decrease, and will de-repress, or induce, uptake of N and vice versa (Padgett and Leonard, 1996). Until recently the evidence for amino acids exerting a regulatory effect on NO3– influx and/or net uptake has been equivocal, several studies indicating significant inhibition of NO3– transport by increased concentrations of several amino acids (Breteler and Arnozis, 1985; Muller and Touraine, 1992; Padgett and Leonard, 1996), but others showing behaviour inconsistent with this scheme (Lee et al., 1992; Tillard et al., 1998). However, there is increasing evidence for a direct effect of amino acids, and most notably of glutamine, on transcript abundance of NRT2 (Glass et al., 2002). When this is taken in conjunction with evidence that diurnal patterns of NO3– uptake are correlated with those for transcript abundance of inducible high affinity transporter coding NRT2 genes (Lejay et al., 1999; Matt et al., 2001), it suggests that the diurnal variation in the vascular amino acid flux may play a crucial role in determining the diurnal variation in NO3– influx.
The present study indicated a transient surge in the xylem concentration (hence flux) of amino acid-N between 6–8 h of the light period and preceding the decrease in net uptake of NO3– between 8–10 h of the light period (Fig. 7). In cereal species over 60% of the xylem amino-N flux may be recycled via the phloem (Cooper and Clarkson, 1989) and if, as seems likely, the proportion is similar or higher in P. pratense, then the xylem amino acid flux should largely reflect the phloem flux of amino acids, subject to a lag period and any modification arising from the exchange (consumption/production) of amino acids in the roots. This assumption is more robust if NO3– reduction occurs predominantly in the leaves, and the high estimated xylem flux of NO3– during the light period indicated this was likely for P. pratense.
The rate of amino acid cycling between xylem–phloem and hence the lag time between the putative sharp increase in phloem amino acid flux and the appearance of its ‘foot-print’ in the xylem were not measured in this study. However, in split-root experiments, significant cycling of 15N between donor and receiver roots was detected 1 h after labelling (Cooper and Clarkson, 1989) and, in the present study, significant translocation of 15N to the shoot was detected within 10 min of its uptake by the roots. Assuming a lag of 0.5 h, then the surge in phloem amino acid-N flux occurred between 0.5–1.5 h prior to the maximum net uptake rate of NO3– (Fig. 7). Hence it did not appear to exert an immediate down-regulation of influx, suggesting it was more likely to act via altered turnover of transporters than by direct allosteric regulation of functional transporters.
The sharp increase in vascular amino acid flux probably resulted from high rates of NO3– assimilation in the shoot, producing amino acids in excess of the shoot’s concurrent capacity for protein synthesis or storage in vacuoles. This interpretation is supported by the sharp increase in the estimated xylem NO3– flux 2 h earlier and by the diurnal variation in N assimilatory activity measured in other species. Both in vitro and in vivo NRA in leaves of Hordeum vulgare L. are maximal close to the middle of artificial photoperiods (Lillo, 1983), and the rate of NO3– assimilation in leaves of Nicotiana tabacum greatly exceeds (x2) the rate of NO3– uptake and translocation during the first part of the light period (Matt et al., 2001). A pivotal role for glutamine in the regulation of N transporter transcript abundance has been suggested (Glass et al., 2002), and it is significant that the surge in xylem amino acid flux measured in the present study was, in large part, accounted for by glutamine.
The observation that 80% of the xylem N fraction was in the form of NO3– throughout the dark period suggests that relatively little additional dark assimilation of NO3– occurred in the roots of this species. Further, the low amino acid concentrations in xylem exudate during darkness imply that the concentration of amino acids in the roots remained relatively low and hence that amino acids levels were unlikely to account for down-regulation of NO3– influx during this period. Other studies have indicated that total amino acid levels in the roots are lowest during the dark period (Pearson and Steer, 1977) or show very little diurnal variation (Matt et al., 2001).
Regulation of diurnal variation in NO3– uptake
At a physiological level, diurnal variation in NO3– uptake has been variously attributed to changes in (1) rates of transpiration and translocation of transport-regulating metabolites (Deane-Drummond, 1990; Le Bot and Kirkby, 1992; Delhon et al., 1995a), (2) carbohydrate availability and energy status in the roots (Hansen, 1980; Gordon et al., 1982; Delhon et al., 1996; Matt et al., 2001), and (3) nutrient demand associated with diurnal growth rhythms in leaf mass and extension rates (Christ, 1978; Schnyder and Nelson, 1988). These mechanisms are not mutually exclusive; their significance probably varies with species, environmental conditions and phase of the diurnal cycle. Further, particular physiological stresses and environmental perturbation may disrupt regulation so that uptake is transiently limited (as opposed to regulated) by an entirely different factor. For example, low carbohydrate status of roots might become directly limiting via NRT2 transcript levels (Matt et al., 2001) towards the end of a long dark period and override post-transcription regulation by NO3– or N assimilate levels. Similarly, drought-induced changes in transpiration might disrupt internal fluxes of regulatory metabolites in the vascular system.
When hourly net uptake rates of NO3– by P. pratense are expressed relative to the maximum rate measured over the diurnal cycle in the current study, five phases of near-constant relative acceleration/deceleration in uptake are apparent (denoted by A, B, C, D, E in Fig. 7), the first two occurring during the light period. With the exception of phase E, these may be explained according to a simple model in which NO3– influx is co-regulated by NO3– and amino acid levels in the cytoplasmic compartment of the roots, assuming (i) NO3– levels in this compartment are determined by the difference between influx and fluxes into the vacuolar compartment and the xylem, (ii) amino acid levels are determined by the difference between the two vascular fluxes and the flux into protein synthesis within the roots, and (iii) assimilation of NO3– in the roots is negligible.
According to this model, diurnal regulation of influx is effected by NO3– levels throughout the dark period (down-regulation) and the first half of the light period (up-regulation), but by amino acids levels during the second half of the light period (down-regulation). The sudden light/dark transitions affect transpiration rate and hence xylem N flux, which in turn affects the concentration of NO3– in the cytoplasmic compartment of the roots, the rate of NO3– assimilation in the shoot and the phloem amino acid flux once the shoot demand for amino acids associated with protein synthesis and vacuolar storage is met.
Whilst this interpretation is undoubtedly over-simplified and lacks specificity in, for example, NO3– transporters, it provides a framework for considering diurnal co-regulation of NO3– uptake by both amino acid and NO3– levels in species assimilating NO3– predominately in the shoots.
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Acknowledgements |
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We thank Mr M Collison for 15N, and Mr J Cockburn for amino acid analysis, and acknowledge the financial support of the Biotechnology and Biological Sciences Research Council of the United Kingdom, and the Norwegian Crop Research Institute.
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