Journal of Experimental Botany, Vol. 52, No. 354, pp. 113-121,
January 2001
© 2001 Oxford University Press
Original Papers |
Rapid disruption of nitrogen metabolism and nitrate transport in spinach plants deprived of sulphate
IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, UK
Received 7 June 2000; Accepted 21 August 2000
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Abstract |
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Hydroponically grown spinach plants were deprived of an external source of sulphate after an initial period when the S-supply was sufficient. The time-course of events following this treatment was monitored. The first responses were found in the uptake and translocation of
and the uptake of 
. The former declined by approximately 50%, the effect being most significant at higher []ext. while the latter increased 6-fold over a 4 d period. Growth in the absence of external resulted in exhaustion of internal pools, the effect being seen first in roots, then in young leaves and, after a marked delay, in mature leaves. In young leaves, there were dramatic increases in the [] and the content of arginine in the first 2 d of S-deprivation. The concentration of glutamine, the most abundant amino acid in S-sufficient conditions, also more than doubled in S-deficient young leaves. The changes in arginine levels were also found in older leaves, but the change in glutamine level was not seen. Assays of nitrate reductase activity (NRA) and nitrate reductase (NR) mRNA from young leaves of S-replete and S-deprived plants revealed a divergence in activity and content only late in the experiments (between days 4 and 8) when results were expressed on a unit leaf basis. However, there were also time-dependent changes in the protein content that kept the specific activities (NRA:protein and RNA:protein) more or less unchanged. The results imply that the impact of S-deficiency on N-utilization are more sensitively monitored by simple measurements of the chemical composition of young leaves than by measurements of NRA or NR transcript abundance. They also suggest that protein synthesis in young leaves is strongly dependent on a continuous supply of from outside the plant. Key words: Spinach, nitrate, nitrate reductase, sulphate.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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All proteins contain sulphur and nitrogen and it is likely that some metabolic co-ordination is necessary to ensure that the fluxes of N and S through their transport and assimilatory pathways meet the amino acid requirements for protein synthesis. It is already known, from studies of cultured cells, that a deficiency of sulphur represses nitrate reductase activity even in the presence of an adequate supply of nitrate (Reuveny et al., 1980
). Results of this kind are sometimes explained in terms of the accumulation of some repressor metabolite which either interacts directly with an enzyme or which down-regulates the transcription of its gene(s). There has been little progress in firmly identifying such metabolite repressors in higher plants. Even in Neurospora, where regulatory circuits for nitrogen and sulphur metabolism have been well defined (Marzluf, 1993), there is no confident identification of molecules which link the expression of transcription factors in the nucleus and the metabolic events in the cytoplasm.
There have been numerous reports of abnormal accumulations of amino compounds and amines in tissues of nutrient-deficient plants (for review see Rabe, 1990). Generally, the observations were made at a time when the plant had developed visual symptoms of deficiency and, more importantly, growth had been slowed down by the shortage of the nutrient in question. Thus, in Medicago sativa (Adams and Sheard, 1966), Hordeum vulgare (Eppendorfer, 1968) and Lolium perenne (Millard et al., 1985), sulphur-deficiency provoked large accumulations of the transport amino acids, glutamine (gln) and asparagine (asn). Such results were interpreted on the basis of an excess of reduced N being available in slowly-growing stressed plants because protein synthesis is concomitantly slow (Rabe, 1990). In an earlier paper from this laboratory it was shown, however, that gln and asn accumulate specifically in the roots of barley plants during the very early stages of sulphate-deprivation and in conditions where neither protein synthesis nor root growth were diminished (Karmoker et al., 1991). In these barley plants it was also shown that the transport of nitrate from the external medium into the roots, and from the roots to the leaves, was markedly inhibited. This raised the possibility that the activity of the nitrate transporter could be modulated by sulphur nutrition.
In the present work spinach plants have been used to explore further the early steps in the perturbation of nitrate transport and nitrogen metabolism during S-deprivation. The results show that NR activity declines in young leaves while nitrate, arginine and glutamine increase greatly. These changes occur more slowly in older leaves and are absent in plants growing in light-limited conditions. During S-deprivation the total RNA of young leaves decreased; if the abundance of NR mRNA was expressed based on its proportion of the total RNA, S-deprivation was without specific effect. However, the absolute abundance of NR mRNA did decrease in parallel with the biochemical measurements of NRA.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Plant culture
Seeds of spinach (Spinacea oleracea cv. Mediana) were germinated on layers of wet filter paper in darkness at 25 °C. After 48–72 h the seedlings were transferred to cylindrical support cups with a suitable mesh screening material at their base. These cups were supported in a lid that was suspended over a PVC bath containing approximately 50 l of culture solution (0.5 strength Letcombe solution: Drew and Saker, 1984
). The radicles were pushed through the mesh so that the roots were in the solution and the etiolated hypocotyls were supported by a bed of black polyethylene beads; these also excluded light from the underlying solution. The culture tanks were located in a temperature-controlled greenhouse (20/17 °C day/night). Temperature control was achieved by heating the greenhouse chamber if the temperature fell below, or by forced ventilation if the temperature rose above that desired. On rare occasions during warm, sunny days where the external air temperature was above that required inside the chamber, the greenhouse temperature would have risen to that of the outside air temperature. Supplementary lighting provided a minimum PAR of 150 µmol m-2 s-1 during low light or darkness to give a 16/8 h day/night cycle, or in a controlled environment chamber (16/8 h day/night, 450 µmol m-2 s-1; 20 °C constant temperature; 60% relative humidity). All plants were grown for 5 weeks prior to the start of each experiment at which time the nutrient solution was changed. Control plants remained in normal 0.5 strength Letcombe solution. MgSO4 was replaced with MgCl2 to impose sulphate deprivation.
Sampling plant material for NR expression and nitrate reductase assays
Two plants were harvested from control and -S treatments at each timepoint. Shoot material was divided into young expanding leaves and older, fully expanded leaves with petioles removed before analysis. Roots were washed in deionized water and blotted dry with paper towels.
Thus there were six samples per treatment per timepoint. Samples were ground in liquid N2 and stored at -70 °C until required. Separate aliquots of each sample were used for both RNA analysis and NR assays.
RNA isolation and Northern analysis
RNA was extracted in duplicate from each sample using 1 g of material by the method described previously (Chattopadhyay et al., 1993). Small quantities of DNA were found in the extracts after the ethanol precipitation step that were removed by the addition of an equal volume of 4 M LiCl, incubation overnight at 4 °C and centrifugation at 12 000 g for 10 min. The RNA pellet was washed with 80% ethanol and resuspended in TE pH 7.5. RNA concentrations were estimated spectrophotometrically at 260 nm in triplicate. For Northern blots, 10 µg RNA was denatured using glyoxal and electrophoresed in agarose (1%) gels using a BES buffer (Grundemann and Koepsaell, 1994) and transferred to Zetaprobe GT membranes by capillary transfer in 10xSSC. The RNA was fixed by baking for 2 h at 80 °C. To visualize the RNA, filters were stained with methylene blue as described earlier (Sambrook et al., 1989) immediately prior to hybridization. Filters were hybridized overnight with 32P-labelled spinach NR cDNA insert (Prosser and Lazarus, 1990) or mitochondrial ß-ATPase (Boutry and Chua, 1986) prepared by random prime labelling (T7QuickPrime, Pharmacia), in 250 mM Na2HPO4 pH 7.2 and 7% SDS at 65 °C and 60 °C, respectively. Washing was performed at 65 °C or 60 °C for 10 min in each of the following solutions: (a) 20 mM Na2 HPO4/5% SDS, (b) 2 SSC/1% SDS, (c) 1 SSC/0.1% SDS. Blots were subjected to autoradiography at -70 °C.
NR extraction and assay
Each sample (0.5 g) was extracted in duplicate in 2 ml extraction buffer consisting of 50 mM MOPS–NaOH pH 7.5, 10 mM MgCl2, 1 mM EDTA, 5 mM dithiothreitol, and 0.1% (v/v) Triton X-100. (Huber et al., 1992) in a chilled mortar. The extracts were centrifuged at 20 000 g for 2 min and the supernatants assayed immediately. The reaction mixtures were as described previously (Huber et al., 1992). The assay volume was 0.5 ml to which 10 µl leaf extract or 50 µl root extract was added. Assays were conducted at 27 °C. The reaction was stopped after 20 min by the addition of 0.5 ml each of 1% sulphanilamide in 3 N HCl and 0.02% naphthyl ethylenediamine dihydrochloride (Wray and Filner, 1970) and the absorbance at 540 nm was determined.
Anion analysis
Freeze-dried, ground plant material (approximately 25 mg) was treated with 160 vols (4 ml) of distilled water. The preparation was raised to boiling point in a microwave and then left at 60 °C for 2 h. The plant material was removed by centrifugation. Anions were separated by HPLC using an isocratic Na2 CO3/NaHCO3 eluent (1.8 mM/1.7 mM), flow rate 2 ml min-1. A sample volume of 20 µl was injected via an auto injector (Spectra-Physics SP8780-XR) into a Dionex AG4–SC guard and AS9–SC analytical columns, coupled to an ASRS-Ultra Anion self-regenerating suppresser. The ions were detected using a conductivity detector (CDM-3, Dionex, UK) and the peak areas were quantified using a Grams/386 Data Acquisition system (Galactic Industries, USA).
Amino acid analysis
The procedure for free amino acid analysis has been described in detail (Clarkson et al., 1989).
Fresh or frozen samples of plant material were mixed with 10 ml of extraction solution (60% (0.5 g)MeOH, 25% chloroform, 15% H2O) and left overnight at -20 °C. Solid material was removed by filtration. Then 1.5 ml of chloroform was added to 5 ml of the extract, mixed thoroughly and finally 1 ml of H2O was added. The extracts were centrifuged at 4000 g for 4 min to separate the phases. The aqueous phase was recovered and for each sample 2 ml aliquots were evaporated to dryness in a freeze drier. The dried residue was redissolved in 2–4 ml of water and 2.5 mmol norleucine was added. After filtration through a 0.22 µm Millipore filter the amino acids were separated by high-performance liquid chromatography (HPLC) (Dionex BIO LC, Amino-Pac PAl amino acid analytical column), using a 50 µl injection, a sodium buffer gradient with ninhydrin post-column derivatization (Trione reagent) and the absorbance at 570 nm was monitored. Norleucine was used as an internal standard to correct for any changes in retention time or sensitivity.
Analysis of 15 uptake and translocation
The basic method used for 15 uptake is described earlier (Karmoker et al., 1991). Spinach plants were transferred from the hydroponic tanks to 1.0 l beakers containing 5 mM 15 (12 atoms% 15N). The solution was aerated for the duration of the uptake period. After 2 h, the roots and shoots were separated. The roots were washed twice in 5.0 l of water (total time 2 min) and excess water removed. The fresh weight of the material was determined. The shoots and roots were then dried in an oven at 60–70 °C, the dry weights were noted and the material was milled. Aliquots of 3–4 mg of the powder were carefully weighed into small tin cups, which were then sealed and placed on the sample changer of an elemental N analyser (Carlo Erber 1500 combuster) attached to an Europa Scientific Tracermass stable isotope mass spectrometer (Clarkson et al., 1989).
Uptake of 35S- and radioassay
The uptake solution contained 1.85x106 Bq 35S- in 1.0 l of 0.75 mM carrier. An aliquot (0.5 ml) of the aqueous extract of freeze-dried material was mixed with 3 ml of scintillation fluid (Optiphase HiSafe, Fisons Chemicals) and ß-emissions recorded on a scintillation counter (Rackbeta, LKB). A sample of the uptake solution was used for calibration.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Effects of sulphate deprivation on the uptake of nitrate and sulphate
After the external
supply had been removed for 4 d, the net uptake of from three concentrations was lower than in +S controls. The plants were pretreated with the 5 mM concentrations and transferred directly to 15N-labelled solution. This may account partially for the effect of deprivation being more significant at the highest concentration of . The effect became more significant at the highest []. At the two higher concentrations, transport of 15N to the shoot could be measured reliably. The –S treatment lowered the delivery of 15N to the shoot by 60% and 42% from solutions containing 0.5 and 5.0 mM , respectively (Table 1).
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In a further experiment, the effect of duration of S-deprivation on uptake of 15N from 5 mM
, and of 35 from 0.75 mM was measured. The results show that sulphate uptake responded rapidly to S-deprivation, increasing 3-fold over 2 d and continuing to rise (Table 2). The uptake of nitrate declined rapidly to a value around 70% of the control, but did not decline further between days 2 and 6. The translocation of nitrate did, however, continue to decline, both as a percentage of the total uptake and in the actual amount of 15N translocated to the shoot.
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Effects on the composition of leaves and roots
In plants that had been growing at a light intensity of approximately 450 µmol m-2 s-1 PAR, there were striking changes in chemical composition that could be detected at an early stage of S-deprivation.
Predictably, the [] in tissues decreased, but at different rates in plant parts; the decline was fastest in roots, where it fell to about 38% of the control after 1 d, and slowest in mature leaves where significant depletion of was not seen until day 3. At the end of a 6 d period of S-deprivation no could be detected in roots, and only traces could be found in young leaves, while in the mature leaves a concentration of greater than 0.5 µmol g-1 FW remained (Fig. 1).
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), –S (
).After 2 d S-deprivation, the [
] of young leaves was nearly double that of the +S control and it continued to rise throughout the experiment. S-deprivation had no effect on the [] in roots. No significant effect on [] was seen in mature leaves until day 4, but by the end of the experiment it had increased to approximately 4-fold that of the control (Fig. 2).
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There were very striking proportional increases in [arg] over a similar time-course. In young leaves the concentration more than doubled over the first 2 d (note log scale in Figs 3
, 4a) while there had not yet been an effect on the mature leaves. By the end of the experiment the [arg] was 40-fold greater in -S young leaves and about 15-fold greater in -S mature ones (Fig. 4a). The pattern of change in [arg] in roots closely resembled that in mature leaves, the principal rise occurring between day 4 and day 8 (data not shown). On day 0 the most abundant amino compound in leaves was glutamine, gln. In mature leaves its concentration did not change while in young leaves it rose steadily during S-deprivation, becoming >4-fold greater than in the +S controls (Fig. 4b). In roots no change in [gln] was measured until day 8, when the concentration became approximately double that of the control. It is interesting to note that there were no systematic effects of sulphate deprivation on the relative amounts of aspartate or glutamate in young leaves; a general trend appeared for their levels to decrease relative to the +S controls.
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When plants growing in the same cabinet were shaded, so that the PAR was less than 60 µmol m-2 s-1 none of the above changes were seen in the slowly growing, light-limited plants. There was a steady decline in the concentrations of all amino acids with time in both +S and -S leaves, and no treatment effect (data not shown).
Nitrate reductase activity, protein and RNA content
The results presented are from plants grown under greenhouse conditions in the summer, but essentially the same results were observed in controlled environment chambers. During the early stages of the experiments, no major differences were observed in NRA, RNA content or protein levels between control and starved plants in any of the tissues (data not shown). However, between 4–8 d after removal, NRA, protein and RNA content show considerable divergence between young leaves of control and starved plants (Fig. 5a–c.). This was not observed in older leaves (Fig. 5d–f), while the decline in NRA and protein content after 4 d was checked and remained at the same levels on day 8. In roots the relatively low levels of NRA, RNA and protein content did not change markedly with treatment or time (data not shown). When nitrate reductase was assayed in the presence or absence of Mg2+, the relative level of activity was similar in control and starved plants (
60% less activity with Mg2+). In shaded plants (<60 µmol m-2 s-1) NRA was 2–3-fold lower, but no differences were observed between control and S-starved plants in any tissues (data not shown).
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Northern blot analysis
When equal quantities of RNA were probed with the NR and ATPase cDNA probes, no noteworthy differences in the level of expression of the respective mRNAs were observed (Fig. 6), minor fluctuations normally mirroring slight differences observed in the NRA of the individual plants. Since the RNA content of the of S-starved young leaves was 50% that of controls, the actual amount of NR mRNA on a fresh weight basis would also be approximately 50% of the controls and would therefore suggest a decrease in NR gene expression (and ATPase) in real terms.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Following a sudden withdrawal of the sulphate supply there in no immediate effect on plant growth (data not shown). It is obvious that the internal pools of sulphur will become diluted as growth continues. In the present results it was seen that the concentration of
declined rapidly in roots, which exported or reduced the sulphate they contained, and in young leaves which would have assimilated the sulphate they contained, while in mature leaves the [] declined more slowly and as much as 30% of the original content was present after 6 d. These mature leaves also continue to expand in the early part of the experiment, but more slowly than young leaves. Similar results were reported for spinach plants deprived of S for 14 d (Dietz, 1989) with the oldest leaves still containing a measurable amount of . This pattern of events fits well with the notion that sulphate may be sequestered in older leaf vacuoles. The rate constant for efflux of from leaf vacuoles of Macroptilium atropurpureum cv. Siratro was 5–10 times lower than for root vacuoles (Bell et al., 1995). It was this, rather than any restriction on re-distribution in the phloem, which made younger leaves so susceptible to short-term effects of S-deprivation. It has been shown that there can be a considerable import/export traffic of labelled in older leaves, but this must involve material in the cytoplasmic pool, rather than in the vacuole (Sunarpi and Anderson, 1996). What happens to the [] in an organ gives a clue to what happens subsequently. Prolonged (2 weeks) S-starvation of spinach plants produced the same overall changes (Dietz, 1989) being most severe in the youngest leaves. In roots the activity of the sulphate transporter increased during the early phase of root depletion; similar observations have been made in many species (Smith et al., 1995: Clarkson et al., 1983; Hawkesford et al., 1993) and it now seems likely that genes encoding the high affinity transporter are strongly up-regulated in this process (Smith et al., 1997).
The most striking effects in leaves were related to an evident dislocation of nitrate assimilation and protein synthesis. Despite a reduced delivery of 15N from the roots to the xylem, most of which is likely to have been (Table 1
), began to accumulate in young leaves from day 2 onwards. The same process occurred in mature leaves, but its onset was delayed and its impact on leaf [] was less. At the same time there was a marked build-up of arginine and glutamine indicating that, in addition, amino acid utilization in protein synthesis was disturbed. This process was quantitatively less in mature leaves. Neither the RNA nor the protein content of S-deprived young leaves increased during this period as it did in the control plants. It is customary to express the abundance of an mRNA relative to the RNA found in a tissue sample; similarly, the activity of an enzyme is expressed on the basis of unit protein. If these conventions are applied, the specific changes in the abundance of the NR mRNA and in the NRA of young leaves are unimpressive. Expressed on a tissue weight basis, however, it becomes clear that the accumulation of in leaves is correlated with a lowered expression of the NR gene and of the NRA (Figs 2; 5a–c). This lowered expression is not necessarily due to the specific repression of the NR, but may reflect a general reduction in protein synthesis caused by a shortage of sulphur amino acids. In its turn, this may lead to a non-specific decline in RNA content. The simple chemical analysis of the leaf tissue seems to be the more sensitive way of measuring the impact of S-deprivation on N-utilization. Among other things, these conclusions point to the delicate poise between protein synthesis and the inorganic sulphur supply, a point made much earlier in experiments with cultured cells (Reuveny et al., 1980).
From a practical viewpoint, this work emphasizes how important it is to maintain an adequate S-supply to a rapidly growing leaf crop, such as spinach. In our experiments the [] in young leaves rose steeply between 4–8 d and showed no signs of flattening out. The results imply that the levels could reach the threshold where the crop is unsaleable because of human dietary considerations (Forman et al., 1985; Van Diest, 1986). Nitrate concentrations of 50 mM or greater are also found when spinach is grown at low light intensity in winter in glass houses (Steingröver et al., 1982). It was shown that in vacuoles of leaves served as an osmoticum, replacing C-compounds that were in short supply because of limited photosynthesis. In this study's work with spinach the effect of S-deprivation on photosynthesis has not been explored, but it is relevant to note that, after 4–6 d, the younger leaves appeared somewhat paler green than the +S controls. In wheat, it was found that net assimilation rates and the content of Rubisco fall markedly in young leaves after 3–6 d S-deprivation (Gilbert et al., 1997). There are many other cases, particularly in the Brassicacea, where sub-optimal S-nutrition results in huge accumulations of nitrate (Schnug, 1990; McGrath and Zhao, 1996). The accumulation of amino acids in sugar beet is known to affect the sugar extraction process adversely. Arginine and glutamine content dramatically increased in the leaves of hydroponically grown S-deficient plants, but a significant increase was found also in field-grown beet where levels of S were low (Sexton et al., 1993). The early identification of S-deficiency has implications for the quality of crops. Its early diagnosis is most important since S-deficiency can be ameliorated by the addition of fertilizer S.
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Notes |
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1 To whom correspondence should be addressed. Fax: +44 1275 394281. E-mail: ian.prosser{at}bbsrc.ac.uk
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