Planta 9 by Springer-Verlag 1977
Planta 137, 107-112 (1977)
Daily Changes in Nitrate Uptake and Metabolism in Capsicum annuum Craig J. Pearson 1 and Barrie T. Steer ~ Department of Agronomy and HorticuIturai Science, University of Sydney, N.S.W. 2006, Australia and 2 Division of Irrigation Research, CSTRO Oriffith, N.S.W, 2680, Australia
Abstract. The diurnal pattern of nitrate uptake by Capsicum annuum L. cv. California Wonder in a con-
stant environment is described by a Fourier harmonic, with the m a x i m u m uptake in the middle of the photoperiod and the m i n i m u m in the middle of the dark period. Comparison of the uptake pattern with that of nitrate reductase (EC 1.6.6.1.) activity suggests against a direct control of one process by the other. This was confirmed by the observation that the pattern of nitrate reductase activity was not altered by restricting nitrate uptake to one h o u r per day. Translocation of lSN from the roots is much greater in the lightperiod than in the dark period. Reduction of 15N in the leaves occurs in the lightperiod but very little is reduced in the dark period. A m i n o acid levels showed marked daily fluctuations but in the roots neither amino adds, sucrose, fructose, glucose nor malate showed fluctuations. The amino acid composition of roots and leaves differed : glutamine + glutamate were relatively m o r e important in leaves than in roots whereas alanine was a more important constituent of roots than of leaves. Key words: A m i n o acids
-
Capsicum
-
Nitrate
uptake -- Rhythmicity (daily).
Introduction
Studies of the daily patterns of regulation of carbon and nitrogen metabolism in plants have described changes in rates of leaf photosynthesis (e.g. Pearson, 1974), the movement of carbon into nitrogen-containing compounds (Noguchi and Tanaki, 1962) and some control points involved (e.g., Steer, 1973, 1974a, b), Abbreviation: NR=nitrate reductase
carbon translocation (Pearson, 1974), levels of carbon and nitrogen in the phloem (Sharkey and Pate, 1976) and nitrogen fixation in nodules (e.g. Minchin and Pate, 1974; Shaposbnikov et al., I976), Manipulative experiments indicate that the availability of nitrogen may affect aspects of carbon metabolism (Avdeeva and Andreeva, 1973; Andreeva et al., 1975) and recently it was suggested that carbon availability to the root may partially control uptake of nitrogen (Ben Zioni et al., 1971 ; Jackson et al., 1976a). We too believe carbon and nitrogen metabolism are interdependent. Nonetheless, we are not aware of any comprehensive studies of daily changes in nitrogen uptake and current models of the regulation of leaf carbon metabolism ignore nitrogen movement. This paper seeks to assist the understanding of daily regulation of carbon and nitrogen metabolism by describing the daily course of nitrogen utilization in Capsicum annuum.
Materials and Methods Capsicum annuum L. cv. California Wonder were grown in 1:1 vermiculite:perlite irrigated daily with a complete nutrient solution containing 6 mmol 1- ~nitrate, in a glasshouse under photoperiods of 13-14 h. At 60 days from sowing, plants selected for uniformity were washed free of rooting medium, rinsed with nutrient solution and transferred to 11.5 cm diam. glazed clay pots lined with polythene bags. The pots were filled with 1 litre of continuously-aerated half-strength nutrient solution containing 0.5 mmol 1-1 nitrate. These plants were placed in a controlled environment cabinet (68 nE cm- 2s- 1, 13.5 h days, 27 • 2C continuous) and allowed 1 day to acclimatise before the experiments. Daily nitrate uptake was measured in three experiments each having 2-4 plants, preconditioned as previously described, which were placed in aerated solution of 2 mmo[1 ~ potassium nitrate in distilled water. Uptake was measured as depletion of nitrate by hourly withdrawing and immediately freezing, 1 ml of solution and subsequently assaying nitrate using an autoanalyzer technique similar to Henzell et al. (1968). The volume of potassium nitrate solution was maintained by addition of water and the entire solu-
108
C.J. Pearson and B.T. Steer: Daily Changes in Nitrate Uptake
tion replaced every 4-5 h. In two experiments the data are presented as hourly values; in an experiment sampling over 32 h, mean hourly values were derived for each 4 5 h period owing to greater variability between samples. Nitrate uptake, translocation and reduction were estimated by placing four preconditioned plants in half-strength nutrient solution containing 0.45 mmol1-1 [lSN]nitrate (30.2% atom excess in calcium nitrate) for 1 h at four times during the day. Exposure to lSN began at the start of the photoperiod (daylength: 13.5 h), after 5.5 h ("mid-photoperiod"), 0.5 h before lights off ("endphotoperiod') and 5 h after lights off ("mid dark period"). At the end of the 1 h exposure all plants were rinsed and returned to another half-strength nutrient solution containing 0.5 mmol 1 1 [14N]nitrate. Plants were sampled at the end of the exposure to lSN and after 5 h in ~r Samples were rinsed for ca. 3 rain in water, separated into roots, stem plus petioles, and leaves. Root and stem were oven-dried at 75 ~ C. The leaves were immediately boiled in 80% ethanol, later in water and separated through Dowex 50-H § 200-400 mesh cation exchange resin into extracts containing amino acids, other soluble N (hereafter called N O ; ) and ethanolinsoluble material. Total nitrogen in each fraction was determined by micro-Kjeldahl technique and percentage ~SN enrichment by standard methods of mass spectrometry. Concentrations of amino acids in roots and leaf number 9 from other plants were measured on a Technicon TSM amino acid analyzer, carbohydrates and malate were assayed by gas chromatography and the spectrofluorimetric method of Snell and Snell (1965) respectively. Fresh weights of the roots, stem and leaves were (mean • standard error) 146+7.2, 67.9• 1.3 and 90.6• g respectively; dry weights were 7.9, 12.2 and 11.0% of fresh weights for roots, stems and leaves respectively. The influence of nitrate availability on nitrate reductase (NR, EC 1.6.6.1.) activity was examined in another experiment by restricting the supply of nitrate in an otherwise complete nutrient solution to 10-11 h after the start of the photoperiod. At the end of the hour the system was flushed with water and the N-free medium recirculated for 23 h. Plants were grown in a flowing liquid culture system for 10 days. They looked healthy in all respects but their leaf nitrate content was low: equivalent to plants grown on 0.6 mmol 1 1 nitrate (Steer, 1974b). NR in single leaves was assayed throughout the 13 h photoperiod using the same technique as in previous work (Steer, 1974b).
Results
Nitrate Uptake There were daily patterns of nitrate uptake with peak rates always occurring in the middle of the photoperiod (Fig. 1). The patterns were characteristic of freerunning cycles and were described by a Fourier harmonic of the form: y = 6.98 - 1.48 cos (~b0 + 6.03 sin (~bt)
(VR=24.2, n=89) where y is btg NO 3 h-* per g whole-plant fw, t is time (h after start of photoperiod) and the periodicity 2~ of the harmonic is 24 h, i.e. (/)= ~ . VR and n are the variance ratio and number of observations respectively. The harmonic accounted for 70.8% of the timeof-day variation.
6
~T J: mn :L2
4
#o #
i
l
l
l
l
J
l
l
l
l
l
l
l
.
.
.
.
.
.
z 22
.
.
.
.
.
b
i
a:
I ~ I 8I I I~3 1 II2 I IZ I ~I8 I ~ h AFTER START OF PHOTOPERIOD
1 2b I 22 1 6
Fig. 1 a and b. Daily nitrate uptake per g whole plant fresh weight. Values in a were determined in 3 experiments (9 e, - ) and the harmonic line fitted for 2 experiments (' O' data omitted). Each point is the mean from 2-4 plant samples. Dark period is indicated by black line on abscissa, h (lower) shows daily nitrate uptake in lSN experiment. Each point is the mean from 2 plants
There were no discontinuities in nitrate uptake at the beginning or end of the photoperiod. Moreover, there was no evidence of secondary peaks in uptake about the commencement of the photoperiod and dark period, as found in in vitro nitrate reductase activities and amino acid labelling from 1r in leaves (Steer, 1974b, 1976). The t SN experiment (Fig. 1 b) gives a measure of absolute nitrate uptake whereas the t4N experiments (Fig. 1a) give an estimate of net uptake. Recent experiments reported by Jackson et al. (1976b) demonstrate a considerable efflux of nitrate from wheat roots at the same time as nitrate uptake. The differences in uptake rates between Figures 1 a and b and the difference in curve amplitude (amplitude--mean was 0.56 in the ISN experiment and 1.3 in the 1r experiment) suggest that nitrate efflux occurs from Capsicum roots. Although rates of uptake are presented on a fresh weight basis to allow comparisons between experiments, dry weight appeared to be a slightly better basis since in the 15N experiment the coefficient of variation was less with dry weight (8.2%) than with fresh weight (12.2%).
109
C.J. Pearson and B.T. Steer: Daily Changes in Nitrate Uptake Table 1. Percentage of uptake 15N which was translocated and reduced to 15N-amino acids and insoluble c o m p o u n d s Photoperiod
Dark Period
StartARer
Mid-
End-
lh
6h
lh
6h
lh
6h
lh
6h
Root Stem Leaves
89.8 6_5 3.5
68.6 20 11
78.6 12 10
61.5 20 18.5
89,0 8 3
86.4 10.5 3
8Z 1 8 4,8
87.1 I0 2.8
Total translocated
10
31
22
38,5
11
13.5
12,8
12.8
22.4
17.6
21.4
8.7
11.4
10.2
11.4
0.88 16.2
0.23 2.1
Translocated without change
7.5
Translocated and reduced to: A m i n o Acids lnsolubles
0.59 2.2
0.78 8.2
At the end of the one hour exposure to l S N NO3, 80--90% of the 1sN within the plant was in the roots (Table 1). In the following 5 h the tSN in the roots remained unchanged in the dark but was depleted at 4% per hour in the light, so that the percentage reaching the leaves was 11 and 18.5 % of the uptake after 6 h in the light. In C. annuum most nitrate reduction occurs in the leaves. NR activity has not been detected in other tissues (Steer, unpublished) and most of the nitrogen in bleeding sap is in the form of nitrate (Steer, 1973). For these reasons, and in the absence of direct measurernent of the chemical form of l SN in the stem samples, we have ascribed all stem 15N to nitrate. Although this assumption may involve some error we believe it to be small. Consequently, the translocared 15N can be subdivided into that which was translocated without change, i.e. stem 15N and leaf thNO3, and that which was reduced in the leaf to 1SN_amino acids and insoluble compounds. The percentage of 15N reduction varied diurnally (Table 1) but the percentage reduced was not closely correlated with total nitrate uptake: linear correlations of reduction and uptake were not significant (P=0.05) after either 1 or 6 h. The percentage of XSN that, once reduced, remained as free amino acids was maintained at a relatively constant value in the light (0.8 _ 0.09%) and at a lower value in the dark (0.23+0.08%) whereas 15N entering the insoluble pools accumulated in the light (Table 1). The flow of nitrogen between the pools calculated from this ~hN study is given in Table 2. The rate of ~hN entry into the shoot was higher during the 0-1 h pulse than in the 1-6 h chase owing to dilution by more recently absorbed I'~N. However, at the end of the pathway the rate of accumulation of insoluble N in the leaf was unchanged during the 6 h in the
0.96 2.9
0.44 1.7
0.06 2.6
0.20 1.3
Table2. Nitrogen flux in o - g N O 3 - N h - 1 per g whole-plant D W for plants of 36.5g DW, between external medium (X), root (R), stem (S) and leaf nitrate (N), amino acid (A) and insolubIe nitrogen (I) pools start-
mid -~
end-photoperiod
dark period
0-t h
X - ~ R 161 R--+S i2.6 S--,N 4,36 N--+A 3.42 A~I 2.70
225 37.8 17.8 7,16 5.38
I68 14.1 3.38 2.95 2.65
128 12.1 2.88 2.47 2.41
1~ h
R--,S S~N N~A A~I
14.2 6.86 6.31 5.98
7.49 0.67 0.56 0.44
2.42 0.37 0,29 0.25
7.68 2.74 2,19 2.00
a Corresponding apparent rate constants for 0-1 kRs 0.68, ksr~ 0.34, kNA 0.016, kAi 0.042 h -1. The root and parent rate constants were obtained by dividing fluxes organ N: thus e.g., if free NO 3 accounted for 10% of the true rate constants would be 6.8 not 0.68 etc.
h were: stem apby total organ N
light, indicating the bulk of the 1sN pulse did not reach the leaf insoluble pool until appreciably after 1 h. The requirement for light for amino acid and insoluble N synthesis, shown in Table I, is also evident from the lower flow rates in the dark in Table 2.
Metabolite Pools
We did not detect daily fluctuations of amino acids within the roots, although there was some suggestion of depletion at the beginning of the dark period (Fig. 2). There were no daily changes in soluble carbohydrates or malate in the roots, the mean
110
C.J. Pearson and B.T. Steer: Daily Changes in Nitrate Uptake
60
In the leaves there were no differences in sucrose o r m a l a t e c o n t e n t s with time o f day. T h e m e a n gg p e r g D W ( n = 12) were sucrose 22.1 + 1.38 a n d m a l a t e 296.6 + 33.3. There was a n i n d i c a t i o n t h a t glucose a n d fructose levels in the leaves were l o w e r in the d a r k ( 0 . 7 9 + 0 . 2 3 gg f r u c t o s e - g - 1 D W ; 0.92_+0.067 gg gluc o s e - g ~ D W ; n = 4 ) t h a n in the light (8.7_+2.5 l~g fructose.g -1Dw; 5.8_+0.67gg glucose-g -~Dw; n - - 8 ) . T h e r e was a m a r k e d daily f l u c t u a t i o n in the a m i n o acid c o n t e n t o f leaves (Fig. 2) in c o n f i r m a t i o n o f earlier d a t a (Steer, 1973). The m a x i m u m c o n t e n t , in the m i d d l e o f the p h o t o p e r i o d , was a b o u t three times less t h a n t h a t r e p o r t e d p r e v i o u s l y (Steer, 1973). This is p r o b a b l y a reflection o f leaf age: the leaves used p r e v i o u s l y were at a b o u t 50% o f full e x p a n s i o n , w h e r e a s in the p r e s e n t e x p e r i m e n t leaves were a t 80% full e x p a n s i o n . W a l l a c e a n d P a t e (1967) r e p o r t e d higher a m i n o a c i d c o n t e n t in y o u n g leaves o f Xanthium pennsylvanicum t h a n in o l d leaves. The a m i n o a c i d c o m p o s i t i o n o f r o o t s a n d leaves are given in T a b l e 3. The c o m p o s i t i o n o f the o r g a n s differed: g l u t a m a t e c o n t r i b u t i n g 23 +_ 2.7% o f the total p o o l in r o o t s b u t in leaves it was a b o u t 6 0 % , except at the e n d o f the p h o t o p e r i o d . Conversely, a l a n i n e was a m a j o r p o o l within the r o o t s b u t was o f less i m p o r t a n c e in leaves. In the leaves at different times o f day, difference o n a % basis (Table 3) lie m a i n l y in a s p a r t a t e , g l u t a m a t e , glycine, a l a n i n e a n d leucine. O n a c o n c e n t r a t i o n basis ( g m o l g - 1 DW), a s p a r t a t e r e m a i n s steady, g l u t a m a t e fluctuates at the same rate as does the t o t a l p o o l , a l a n i n e a n d leucine fluctuate m o r e t h a n the t o t a l p o o l (i.e. are m o r e imp o r t a n t where the p o o l is large in the m i d d l e o f the day) a n d glycine pe~/ks w h e n the t o t a l p o o l is low (end o f p h o t o p e r i o d ) . This i n d e p e n d e n c e o f a s p a r t a t e p o o l size f r o m the f l u c t u a t i o n s in the t o t a l a m i n o acid p o o l was n o t f o u n d in y o u n g e r leaves (Steer, 1973) w h e r e a s there was s o m e i n d i c a t i o n s f r o m y o u n ger leaves ( u n p u b l i s h e d ) o f glycine p o o l size acting i n d e p e n d e n t l y . I n t o b a c c o leaves ( N o g u c h i a n d T a maki, 1962) a s p a r t a t e h a d a m a x i m u m p o o l size at night w h e r e a s o t h e r a m i n o acids h a d p e a k s in the m i d d l e o f the p h o t o p e r i o d .
d
Planta 137, 107-112 (1977)
Daily Changes in Nitrate Uptake and Metabolism in Capsicum annuum Craig J. Pearson 1 and Barrie T. Steer ~ Department of Agronomy and HorticuIturai Science, University of Sydney, N.S.W. 2006, Australia and 2 Division of Irrigation Research, CSTRO Oriffith, N.S.W, 2680, Australia
Abstract. The diurnal pattern of nitrate uptake by Capsicum annuum L. cv. California Wonder in a con-
stant environment is described by a Fourier harmonic, with the m a x i m u m uptake in the middle of the photoperiod and the m i n i m u m in the middle of the dark period. Comparison of the uptake pattern with that of nitrate reductase (EC 1.6.6.1.) activity suggests against a direct control of one process by the other. This was confirmed by the observation that the pattern of nitrate reductase activity was not altered by restricting nitrate uptake to one h o u r per day. Translocation of lSN from the roots is much greater in the lightperiod than in the dark period. Reduction of 15N in the leaves occurs in the lightperiod but very little is reduced in the dark period. A m i n o acid levels showed marked daily fluctuations but in the roots neither amino adds, sucrose, fructose, glucose nor malate showed fluctuations. The amino acid composition of roots and leaves differed : glutamine + glutamate were relatively m o r e important in leaves than in roots whereas alanine was a more important constituent of roots than of leaves. Key words: A m i n o acids
-
Capsicum
-
Nitrate
uptake -- Rhythmicity (daily).
Introduction
Studies of the daily patterns of regulation of carbon and nitrogen metabolism in plants have described changes in rates of leaf photosynthesis (e.g. Pearson, 1974), the movement of carbon into nitrogen-containing compounds (Noguchi and Tanaki, 1962) and some control points involved (e.g., Steer, 1973, 1974a, b), Abbreviation: NR=nitrate reductase
carbon translocation (Pearson, 1974), levels of carbon and nitrogen in the phloem (Sharkey and Pate, 1976) and nitrogen fixation in nodules (e.g. Minchin and Pate, 1974; Shaposbnikov et al., I976), Manipulative experiments indicate that the availability of nitrogen may affect aspects of carbon metabolism (Avdeeva and Andreeva, 1973; Andreeva et al., 1975) and recently it was suggested that carbon availability to the root may partially control uptake of nitrogen (Ben Zioni et al., 1971 ; Jackson et al., 1976a). We too believe carbon and nitrogen metabolism are interdependent. Nonetheless, we are not aware of any comprehensive studies of daily changes in nitrogen uptake and current models of the regulation of leaf carbon metabolism ignore nitrogen movement. This paper seeks to assist the understanding of daily regulation of carbon and nitrogen metabolism by describing the daily course of nitrogen utilization in Capsicum annuum.
Materials and Methods Capsicum annuum L. cv. California Wonder were grown in 1:1 vermiculite:perlite irrigated daily with a complete nutrient solution containing 6 mmol 1- ~nitrate, in a glasshouse under photoperiods of 13-14 h. At 60 days from sowing, plants selected for uniformity were washed free of rooting medium, rinsed with nutrient solution and transferred to 11.5 cm diam. glazed clay pots lined with polythene bags. The pots were filled with 1 litre of continuously-aerated half-strength nutrient solution containing 0.5 mmol 1-1 nitrate. These plants were placed in a controlled environment cabinet (68 nE cm- 2s- 1, 13.5 h days, 27 • 2C continuous) and allowed 1 day to acclimatise before the experiments. Daily nitrate uptake was measured in three experiments each having 2-4 plants, preconditioned as previously described, which were placed in aerated solution of 2 mmo[1 ~ potassium nitrate in distilled water. Uptake was measured as depletion of nitrate by hourly withdrawing and immediately freezing, 1 ml of solution and subsequently assaying nitrate using an autoanalyzer technique similar to Henzell et al. (1968). The volume of potassium nitrate solution was maintained by addition of water and the entire solu-
108
C.J. Pearson and B.T. Steer: Daily Changes in Nitrate Uptake
tion replaced every 4-5 h. In two experiments the data are presented as hourly values; in an experiment sampling over 32 h, mean hourly values were derived for each 4 5 h period owing to greater variability between samples. Nitrate uptake, translocation and reduction were estimated by placing four preconditioned plants in half-strength nutrient solution containing 0.45 mmol1-1 [lSN]nitrate (30.2% atom excess in calcium nitrate) for 1 h at four times during the day. Exposure to lSN began at the start of the photoperiod (daylength: 13.5 h), after 5.5 h ("mid-photoperiod"), 0.5 h before lights off ("endphotoperiod') and 5 h after lights off ("mid dark period"). At the end of the 1 h exposure all plants were rinsed and returned to another half-strength nutrient solution containing 0.5 mmol 1 1 [14N]nitrate. Plants were sampled at the end of the exposure to lSN and after 5 h in ~r Samples were rinsed for ca. 3 rain in water, separated into roots, stem plus petioles, and leaves. Root and stem were oven-dried at 75 ~ C. The leaves were immediately boiled in 80% ethanol, later in water and separated through Dowex 50-H § 200-400 mesh cation exchange resin into extracts containing amino acids, other soluble N (hereafter called N O ; ) and ethanolinsoluble material. Total nitrogen in each fraction was determined by micro-Kjeldahl technique and percentage ~SN enrichment by standard methods of mass spectrometry. Concentrations of amino acids in roots and leaf number 9 from other plants were measured on a Technicon TSM amino acid analyzer, carbohydrates and malate were assayed by gas chromatography and the spectrofluorimetric method of Snell and Snell (1965) respectively. Fresh weights of the roots, stem and leaves were (mean • standard error) 146+7.2, 67.9• 1.3 and 90.6• g respectively; dry weights were 7.9, 12.2 and 11.0% of fresh weights for roots, stems and leaves respectively. The influence of nitrate availability on nitrate reductase (NR, EC 1.6.6.1.) activity was examined in another experiment by restricting the supply of nitrate in an otherwise complete nutrient solution to 10-11 h after the start of the photoperiod. At the end of the hour the system was flushed with water and the N-free medium recirculated for 23 h. Plants were grown in a flowing liquid culture system for 10 days. They looked healthy in all respects but their leaf nitrate content was low: equivalent to plants grown on 0.6 mmol 1 1 nitrate (Steer, 1974b). NR in single leaves was assayed throughout the 13 h photoperiod using the same technique as in previous work (Steer, 1974b).
Results
Nitrate Uptake There were daily patterns of nitrate uptake with peak rates always occurring in the middle of the photoperiod (Fig. 1). The patterns were characteristic of freerunning cycles and were described by a Fourier harmonic of the form: y = 6.98 - 1.48 cos (~b0 + 6.03 sin (~bt)
(VR=24.2, n=89) where y is btg NO 3 h-* per g whole-plant fw, t is time (h after start of photoperiod) and the periodicity 2~ of the harmonic is 24 h, i.e. (/)= ~ . VR and n are the variance ratio and number of observations respectively. The harmonic accounted for 70.8% of the timeof-day variation.
6
~T J: mn :L2
4
#o #
i
l
l
l
l
J
l
l
l
l
l
l
l
.
.
.
.
.
.
z 22
.
.
.
.
.
b
i
a:
I ~ I 8I I I~3 1 II2 I IZ I ~I8 I ~ h AFTER START OF PHOTOPERIOD
1 2b I 22 1 6
Fig. 1 a and b. Daily nitrate uptake per g whole plant fresh weight. Values in a were determined in 3 experiments (9 e, - ) and the harmonic line fitted for 2 experiments (' O' data omitted). Each point is the mean from 2-4 plant samples. Dark period is indicated by black line on abscissa, h (lower) shows daily nitrate uptake in lSN experiment. Each point is the mean from 2 plants
There were no discontinuities in nitrate uptake at the beginning or end of the photoperiod. Moreover, there was no evidence of secondary peaks in uptake about the commencement of the photoperiod and dark period, as found in in vitro nitrate reductase activities and amino acid labelling from 1r in leaves (Steer, 1974b, 1976). The t SN experiment (Fig. 1 b) gives a measure of absolute nitrate uptake whereas the t4N experiments (Fig. 1a) give an estimate of net uptake. Recent experiments reported by Jackson et al. (1976b) demonstrate a considerable efflux of nitrate from wheat roots at the same time as nitrate uptake. The differences in uptake rates between Figures 1 a and b and the difference in curve amplitude (amplitude--mean was 0.56 in the ISN experiment and 1.3 in the 1r experiment) suggest that nitrate efflux occurs from Capsicum roots. Although rates of uptake are presented on a fresh weight basis to allow comparisons between experiments, dry weight appeared to be a slightly better basis since in the 15N experiment the coefficient of variation was less with dry weight (8.2%) than with fresh weight (12.2%).
109
C.J. Pearson and B.T. Steer: Daily Changes in Nitrate Uptake Table 1. Percentage of uptake 15N which was translocated and reduced to 15N-amino acids and insoluble c o m p o u n d s Photoperiod
Dark Period
StartARer
Mid-
End-
lh
6h
lh
6h
lh
6h
lh
6h
Root Stem Leaves
89.8 6_5 3.5
68.6 20 11
78.6 12 10
61.5 20 18.5
89,0 8 3
86.4 10.5 3
8Z 1 8 4,8
87.1 I0 2.8
Total translocated
10
31
22
38,5
11
13.5
12,8
12.8
22.4
17.6
21.4
8.7
11.4
10.2
11.4
0.88 16.2
0.23 2.1
Translocated without change
7.5
Translocated and reduced to: A m i n o Acids lnsolubles
0.59 2.2
0.78 8.2
At the end of the one hour exposure to l S N NO3, 80--90% of the 1sN within the plant was in the roots (Table 1). In the following 5 h the tSN in the roots remained unchanged in the dark but was depleted at 4% per hour in the light, so that the percentage reaching the leaves was 11 and 18.5 % of the uptake after 6 h in the light. In C. annuum most nitrate reduction occurs in the leaves. NR activity has not been detected in other tissues (Steer, unpublished) and most of the nitrogen in bleeding sap is in the form of nitrate (Steer, 1973). For these reasons, and in the absence of direct measurernent of the chemical form of l SN in the stem samples, we have ascribed all stem 15N to nitrate. Although this assumption may involve some error we believe it to be small. Consequently, the translocared 15N can be subdivided into that which was translocated without change, i.e. stem 15N and leaf thNO3, and that which was reduced in the leaf to 1SN_amino acids and insoluble compounds. The percentage of 15N reduction varied diurnally (Table 1) but the percentage reduced was not closely correlated with total nitrate uptake: linear correlations of reduction and uptake were not significant (P=0.05) after either 1 or 6 h. The percentage of XSN that, once reduced, remained as free amino acids was maintained at a relatively constant value in the light (0.8 _ 0.09%) and at a lower value in the dark (0.23+0.08%) whereas 15N entering the insoluble pools accumulated in the light (Table 1). The flow of nitrogen between the pools calculated from this ~hN study is given in Table 2. The rate of ~hN entry into the shoot was higher during the 0-1 h pulse than in the 1-6 h chase owing to dilution by more recently absorbed I'~N. However, at the end of the pathway the rate of accumulation of insoluble N in the leaf was unchanged during the 6 h in the
0.96 2.9
0.44 1.7
0.06 2.6
0.20 1.3
Table2. Nitrogen flux in o - g N O 3 - N h - 1 per g whole-plant D W for plants of 36.5g DW, between external medium (X), root (R), stem (S) and leaf nitrate (N), amino acid (A) and insolubIe nitrogen (I) pools start-
mid -~
end-photoperiod
dark period
0-t h
X - ~ R 161 R--+S i2.6 S--,N 4,36 N--+A 3.42 A~I 2.70
225 37.8 17.8 7,16 5.38
I68 14.1 3.38 2.95 2.65
128 12.1 2.88 2.47 2.41
1~ h
R--,S S~N N~A A~I
14.2 6.86 6.31 5.98
7.49 0.67 0.56 0.44
2.42 0.37 0,29 0.25
7.68 2.74 2,19 2.00
a Corresponding apparent rate constants for 0-1 kRs 0.68, ksr~ 0.34, kNA 0.016, kAi 0.042 h -1. The root and parent rate constants were obtained by dividing fluxes organ N: thus e.g., if free NO 3 accounted for 10% of the true rate constants would be 6.8 not 0.68 etc.
h were: stem apby total organ N
light, indicating the bulk of the 1sN pulse did not reach the leaf insoluble pool until appreciably after 1 h. The requirement for light for amino acid and insoluble N synthesis, shown in Table I, is also evident from the lower flow rates in the dark in Table 2.
Metabolite Pools
We did not detect daily fluctuations of amino acids within the roots, although there was some suggestion of depletion at the beginning of the dark period (Fig. 2). There were no daily changes in soluble carbohydrates or malate in the roots, the mean
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C.J. Pearson and B.T. Steer: Daily Changes in Nitrate Uptake
60
In the leaves there were no differences in sucrose o r m a l a t e c o n t e n t s with time o f day. T h e m e a n gg p e r g D W ( n = 12) were sucrose 22.1 + 1.38 a n d m a l a t e 296.6 + 33.3. There was a n i n d i c a t i o n t h a t glucose a n d fructose levels in the leaves were l o w e r in the d a r k ( 0 . 7 9 + 0 . 2 3 gg f r u c t o s e - g - 1 D W ; 0.92_+0.067 gg gluc o s e - g ~ D W ; n = 4 ) t h a n in the light (8.7_+2.5 l~g fructose.g -1Dw; 5.8_+0.67gg glucose-g -~Dw; n - - 8 ) . T h e r e was a m a r k e d daily f l u c t u a t i o n in the a m i n o acid c o n t e n t o f leaves (Fig. 2) in c o n f i r m a t i o n o f earlier d a t a (Steer, 1973). The m a x i m u m c o n t e n t , in the m i d d l e o f the p h o t o p e r i o d , was a b o u t three times less t h a n t h a t r e p o r t e d p r e v i o u s l y (Steer, 1973). This is p r o b a b l y a reflection o f leaf age: the leaves used p r e v i o u s l y were at a b o u t 50% o f full e x p a n s i o n , w h e r e a s in the p r e s e n t e x p e r i m e n t leaves were a t 80% full e x p a n s i o n . W a l l a c e a n d P a t e (1967) r e p o r t e d higher a m i n o a c i d c o n t e n t in y o u n g leaves o f Xanthium pennsylvanicum t h a n in o l d leaves. The a m i n o a c i d c o m p o s i t i o n o f r o o t s a n d leaves are given in T a b l e 3. The c o m p o s i t i o n o f the o r g a n s differed: g l u t a m a t e c o n t r i b u t i n g 23 +_ 2.7% o f the total p o o l in r o o t s b u t in leaves it was a b o u t 6 0 % , except at the e n d o f the p h o t o p e r i o d . Conversely, a l a n i n e was a m a j o r p o o l within the r o o t s b u t was o f less i m p o r t a n c e in leaves. In the leaves at different times o f day, difference o n a % basis (Table 3) lie m a i n l y in a s p a r t a t e , g l u t a m a t e , glycine, a l a n i n e a n d leucine. O n a c o n c e n t r a t i o n basis ( g m o l g - 1 DW), a s p a r t a t e r e m a i n s steady, g l u t a m a t e fluctuates at the same rate as does the t o t a l p o o l , a l a n i n e a n d leucine fluctuate m o r e t h a n the t o t a l p o o l (i.e. are m o r e imp o r t a n t where the p o o l is large in the m i d d l e o f the day) a n d glycine pe~/ks w h e n the t o t a l p o o l is low (end o f p h o t o p e r i o d ) . This i n d e p e n d e n c e o f a s p a r t a t e p o o l size f r o m the f l u c t u a t i o n s in the t o t a l a m i n o acid p o o l was n o t f o u n d in y o u n g e r leaves (Steer, 1973) w h e r e a s there was s o m e i n d i c a t i o n s f r o m y o u n ger leaves ( u n p u b l i s h e d ) o f glycine p o o l size acting i n d e p e n d e n t l y . I n t o b a c c o leaves ( N o g u c h i a n d T a maki, 1962) a s p a r t a t e h a d a m a x i m u m p o o l size at night w h e r e a s o t h e r a m i n o acids h a d p e a k s in the m i d d l e o f the p h o t o p e r i o d .
d
