Nitrate Uptake, Nitrate Reductase Distribution and ... - Oxford Journals

Annals of Botany 90: 315±323, 2002 doi:10.1093/aob/mcf190, available online at www.aob.oupjournals.org

Nitrate Uptake, Nitrate Reductase Distribution and their Relation to Proton Release in Five Nodulated Grain Legumes X . H . F A N 1 , 2 , C . T A N G 1 , * and Z . R EN G EL 1 of Soil Science and Plant Nutrition, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia and 2Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, P. R. China

1Department

Received: 28 February 2002 Returned for revision: 25 April 2002 Accepted: 27 May 2002 Published electronically: 5 August 2002

Nitrate uptake, nitrate reductase activity (NRA) and net proton release were compared in ®ve grain legumes grown at 0´2 and 2 mM nitrate in nutrient solution. Nitrate treatments, imposed on 22-d-old, fully nodulated plants, lasted for 21 d. Increasing nitrate supply did not signi®cantly in¯uence the growth of any of the species during the treatment, but yellow lupin (Lupinus luteus) had a higher growth rate than the other species examined. At 0´2 mM nitrate supply, nitrate uptake rates ranged from 0´6 to 1´5 mg N g±1 d±1 in the order: yellow lupin > ®eld pea (Pisum sativum) > chickpea (Cicer arietinum) > narrow-leafed lupin (L. angustifolius) > white lupin (L. albus). At 2 mM nitrate supply, nitrate uptake ranged from 1´7 to 8´2 mg N g±1 d±1 in the order: ®eld pea > chickpea > white lupin > yellow lupin > narrow-leafed lupin. Nitrate reductase activity increased with increased nitrate supply, with the majority of NRA being present in shoots. Field pea and chickpea had much higher shoot NRA than the three lupin species. When 0´2 mM nitrate was supplied, narrow-leafed lupin released the most H+ per unit root biomass per day, followed by yellow lupin, white lupin, ®eld pea and chickpea. At 2 mM nitrate, narrow-leafed lupin and yellow lupin showed net proton release, whereas the other species, especially ®eld pea, showed net OH± release. Irrespective of legume species and nitrate supply, proton release was negatively correlated with nitrate uptake and NRA in shoots, but not with NRA in roots. ã 2002 Annals of Botany Company Key words: Distribution, nitrate uptake, nitrate reductase, N2 ®xation, proton release, species variation.

INTRODUCTION The form of nitrogen plays a key role in cation±anion relationships in plants and hence in net proton release by roots. Ammonium nutrition and N2 ®xation are accompanied by the release of H+, whereas NO3± uptake involves OH± excretion (Tang and Rengel, 2002). The acid generated by N2-®xing legumes is reported to range from 0´2 to 1´6 mole H+ per mole N ®xed, whereas the uptake and assimilation of 1 mole NH4+ is associated with the excretion of 1´1 to 1´6 moles of H+ (Raven et al., 1990). When nitrate is entirely assimilated in roots, for every mole of NO3± reduced to NH4+ and assimilated into organic matter, close to 1 mole of OH± ions is produced. If NO3± is assimilated in shoots, depending upon the storage capacity of the shoot for products of malate decarboxylation, the amounts of OH± ions released range from 0 to 1 mole per mole NO3± assimilated (Bolan et al., 1991). Nitrate is generally considered to be the major form of inorganic nitrogen in most agricultural soils (Pilbeam and Kirkby, 1992). Leaching of nitrate, which is produced during N transformations, is a major cause of topsoil acidi®cation (Tang et al., 2000). Thus, prevention of nitrate leaching is an important step towards minimizing soil acidi®cation. Plant species and genotypes, including legumes, differ greatly in their ability to take up soil nitrate (Caba et al., 1993; Armstrong et al., 1994; Tang et al., * For correspondence. Fax +61 8 8380 1050, e-mail cxtang@cyllene. uwa.edu.au

1999), and thus release H+/OH± (Tang et al., 1999). For example, at a ®eld site, the ®eld pea cultivar `Wirrega' took up ten times more soil N than did `Dundale' despite similar amounts of N2 being ®xed by both cultivars (Armstrong et al., 1994). High concentrations of nitrate in soils can impair nodulation and depress N2 ®xation (Harper and Gibson, 1984; Beccana and Sprent 1987; Macduff et al., 1996) but there is also genotypic variation among legumes in sensitivity to nitrate (Harper and Gibson, 1984; Chalifour and Nelson, 1988). Therefore, selecting legumes that have an enhanced ability to take up nitrate with minimum interference to N2 ®xation could be an important strategy for minimizing soil acidi®cation in legume-based agro-ecosystems. Nitrate absorbed by plants must be reduced to ammonium before incorporation into amino acids. Nitrate reduction is catalysed by nitrate reductase and nitrite reductase. There are large differences among species and genotypes in the role of shoot and root systems in the reduction of nitrate (Andrews, 1986; Wallace, 1986; Chalifour and Nelson, 1988). Plants can be categorized into three groups according to the major site of nitrate reduction: (1) root; (2) root and shoot; and (3) shoot (Stewart et al., 1986). However, it is not known whether the site of nitrate reduction is related to the ability of plants to take up nitrate and the release net acid by the root. A previous study examined the effect of nitrate supply on root acid release of grain legumes grown in soil (Tang et al., ã 2002 Annals of Botany Company

316

Fan et al. Ð Nitrate Uptake, Nitrate Reductase and Proton Release in Legumes

1999). The present study compares nitrate uptake and net H+/OH± release, and their relationship with the activity and distribution of nitrate reductase, in nodulated plants of ®ve grain legumes. Plants were grown in nutrient solution to facilitate direct measurements of H+/OH± release and nitrate uptake by the roots.

MATERIALS AND METHODS The experiment included ®ve grain legume species and two nitrate levels (0´2 and 2 mM) in triplicate, and was conducted in a glasshouse with natural light at 20/15 °C (12/12 h). The ®ve grain legume species were chickpea (Cicer arietinum L. `Tyson'), yellow lupin (Lupinus luteus L. `Teo'), white lupin (L. albus L. `Kiev'), narrow-leafed lupin (L. angustifolius L. `Gunguuru') and ®eld pea (Pisum sativum L. `Dundale'). Seeds were germinated on stainless steel screens covered with paper towel suspended over an aerated solution containing 0´6 mM CaCl2 and 5 mM H3BO3 at pH 5´0±5´5 for 5 d. To achieve similar total biomass production, 10±18 uniform seedlings of individual species were then transferred to each pot containing 5´5 l of solution with the following composition of basal nutrients (mM): KH2PO4, 20; K2SO4, 600; MgSO4, 200; CaCl2, 600; H3BO3, 5; Na2MoO4, 0´03; ZnSO4, 0´75; MnSO4, 1´0; CoSO4, 0´2; CuSO4, 0´2; and FeNaEDTA, 10. De-ionized water was used throughout the experiment. A water suspension of nodule bacteria (108 cells per pot) was added to the nutrient solution. The bacterial suspension was a mixture of commercial peat inocula Group Gâ [Bradyrhizobium sp. (Lupinus) WU425] for Lupinus species, Group Nâ [Rhizobium sp. (Cicer) CC1192] for chickpea and Group Eâ (Rhizobium leguminosarum bv. viceae SU303) for ®eld pea. The nutrient solution was not changed for the ®rst 5 d after transplanting, during which the solution pH was adjusted twice daily to the initial pH of around 5´5 to ensure good nodulation. The nutrient solutions were then changed three times a week without further addition of nodule bacteria, and the pH was adjusted daily to 5´5. Two nitrate treatments [0´2 and 2 mM N as Ca(NO3)2] were imposed at 22 d after germination (day 0) when all plants were fully nodulated. An extra set of pots containing treatment solutions without plants was used as the control. The nutrient solution was changed three times a week. Solution pH was adjusted daily to 5´5 using a standardized 0´1 M KOH or 0´1 M HCl. Two plants on day 0, and three to ®ve plants on days 7, 14 and 21 were sub-sampled from each pot. Shoot and root weights were recorded. Relative growth rate (R) was calculated for the treatment period (t2 ± t1 in days) based on initial dry weights (W1) and dry weights at the ®nal harvest (W2) using the following equation: R = (ln W2 ± ln W1)/(t2 ± t1) Samples of the ®nal solution from every pot were retained for determination of NO3± and H+/OH± release each time the nutrient solutions were changed. Samples were frozen if they could not be analysed immediately. The pH of the solutions was measured using an Orion EA940 pH meter

with a combined glass electrode. The amounts of H+/OH± released by plants were calculated from the amounts of KOH or HCl added to adjust the pH during plant growth and the amounts of KOH or HCl used for the back-titration of the ®nal solutions (three from each pot weekly) after plant growth against the control solution without plants (pH around 5´5) (McLay et al., 1997). By using control solutions, the effects of possible variation in nutrient composition and factors other than plants on changes of solution pH and nitrate are minimized. Nitrate concentration in the ®nal solutions was measured using an autoanalyser (Skalar, Delft, The Netherlands). The amount of nitrate depletion in nutrient solution was considered to be the amount of nitrate taken up by plants. Speci®c H+/OH± release or the speci®c absorption rate of nitrate during various growth stages was calculated according to the following equation: S or A = [Htot or Ntot/(W2 ± W1)] 3 [(ln W2 ± ln W1)/(t2 ± t1)] where S is speci®c H+/OH± release, A is the speci®c absorption rate of nitrate, and Htot and Ntot represent total amounts of H+ (negative values for OH±) released and total amounts of nitrate taken up by plants, respectively, between times t1 and t2 with corresponding whole plant dry weights of W1 and W2. The extraction and assay of shoot and root nitrate reductase (NR) were performed following the method described by Langelaan and Troelstra (1992). Brie¯y, duplicate samples of fresh tissues (approx. 0´1 g) were incubated at 30 °C in the dark for 1 h after vacuumin®ltration. For shoot assay of all the species, the buffer contained 0´1 M KNO3, 15 ml l±1 propanol and 0´1 M phosphate buffered at pH 7´5. For the root assay, propanol was not added to the buffer. Nitrite was measured colorimetrically at 540 nm after reacting with N-(1naphthyl)-ethylene-diamine dihydrochloride. Nitrate reductase activity (NRA) was estimated from the amount of nitrite produced. Total NRA in shoots and roots was calculated by multiplying NRA per gram dry weight by the dry weight of each component. The percentage of total plant NRA contributed by each component was calculated by dividing the NRA of each component by total plant NRA. For chemical analysis, samples of three replicate pots were bulked proportionally to total dry weights of individual replicates. Plant total N concentration was determined using a Leco CHN analyser (St Joseph, MI, USA). Concentrations of total plant Ca, Mg, K, Na, P and S were determined using an inductively coupled plasma emission spectrometer (ICPAES) following nitric acid digestion. The concentration of Cl± in water extracts was analysed colorimetrically using an autoanalyser (Lachat, Milwaukee, WI, USA). NonN excess cations were calculated as the sum of charge concentrations of Ca2+, Mg2+, K+ and Na+ minus the sum of SO42± (S), H2PO4± (P) and Cl± (Cl). The amounts of N2 ®xed were calculated as the difference between total plant N and the amounts of nitrate depleted from nutrient solution by plants. The amounts of proton release (Y) were also calculated according to the equation of Troelstra et al. (1985):


317

TA B L E 1. Dry weights of shoots and roots and relative growth rates (R) of ®ve grain legumes grown at 0´2 (N1) and 2 mM (N2) nitrate for various periods Dry weights (g per plant) R (mg g±1 d±1)

Days after nitrate addition 0

7

Species Shoot Chickpea (Cicer arietinum) White lupin (Lupinus albus) Narrow-leafed lupin (L. angustifolius) Field pea (Pisum sativum) Yellow lupin (L. luteus) LSD (P = 0´05) Species Root Chickpea White lupin Narrow-leafed lupin Field pea Yellow lupin LSD (P = 0´05) Species

0´14 0´56 0´17 0´25 0´19

21

Days 0±21

N1

N2

N1

N2

N1

N2

N1

N2

0´18 1´02 0´26 0´52 0´39

0´18 1´03 0´29 0´55 0´37

0´29 1´64 0´48 0´90 0´81

0´40 1´86 0´55 1´11 0´96

0´68 2´69 1´12 1´53 1´91

0´91 3´30 1´39 1´55 1´97

32 32 39 37 46

38 37 44 33 47

0´12 0´12 0´24 0´13 0´14 0´07

14

0´07 0´17 0´46 0´22 0´24 0´20

0´03

0´14 0´14 0´44 0´19 0´20 0´15

0´20 0´77 0´31 0´33 0´45

0´04

0´40 0´21 0´89 0´33 0´31 0´47

0´09

0´48 1´40 0´74 0´42 0´71

7 0´56 1´50 0´84 0´40 0´90

0´16

29 37 36 23 46

32 38 39 24 52 7

All plants were nodulated and received nitrate treatments from 22 d after germination (as day 0). The effect of nitrate supply and the interaction between species and nitrate supply were not signi®cant at P = 0´05.

Y = (C ± A) + 0´946 Norg ± 2x ± y where all parameters are in mmol per plant and (C ± A) represents non-N excess cations, Norg plant organic N (total N was used in this study), and x and y represent amounts of the organic N derived from nitrate and N2 ®xation, respectively. Data were analysed using ANOVA. Means were compared using least signi®cant differences (LSD) at the 5 % probability level. R E SU L T S Plant growth

Shoot dry weights varied markedly among the species, being largest for white lupin at all stages of growth, followed by yellow lupin, ®eld pea, narrow-leafed lupin and chickpea (Table 1). Shoot weights increased exponentially with time. Yellow lupin had the highest relative growth rate during 21 d of nitrate treatment. Relative growth rates were similar to those reported previously for the same species (McLay et al., 1997). Root dry weights followed a similar pattern to shoot weights, with those for white lupin being highest and those for ®eld pea lowest. The dry matter production of both shoots and roots was not signi®cantly affected by nitrate supply, although dry weights on days 14 and 21, and relative growth rates were slightly higher at 2 mM than at 0´2 mM nitrate supply (Table 1). Nitrate uptake

Total nitrate uptake per plant varied signi®cantly among species and increased with increasing nitrate supply and

with time (Table 2). In general, white lupin had the highest and chickpea the lowest nitrate uptake per plant, mainly related to biomass production. Speci®c nitrate absorption showed less variation among species compared with total nitrate uptake (Table 2). At 0´2 mM nitrate supply, speci®c nitrate uptake was highest in yellow lupin and lowest in white lupin during the ®rst 14 d of treatment, and highest in ®eld pea and lowest in white lupin during 14±21 d. With duration of nitrate treatment, speci®c nitrate uptake increased in chickpea and ®eld pea and to a lesser extent in narrow-leafed lupin, but generally remained unchanged in white lupin and yellow lupin. Increasing nitrate supply increased speci®c nitrate uptake in all species. At 2 mM nitrate supply, speci®c nitrate uptake was generally higher in ®eld pea and chickpea than in the lupin species (Table 2). NRA

Prior to addition of nitrate (day 0), NRA was very low in both shoots and roots of all species, with ®eld pea having higher activity than the other species (Table 3). NRA increased dramatically in the presence of nitrate and increased with increasing level of nitrate supply (Table 3). There was a striking difference in NRA in shoots among species. Of the species studied, ®eld pea had the highest NRA in shoots, followed by chickpea, whereas white lupin showed the lowest NRA. NRA in roots did not display the same pattern as that in shoots, with much less variation among species (Table 3). At 2 mM nitrate supply, white lupin had higher NRA in the roots than the other species. Most NRA was located in the shoots even though the distribution of NRA differed among species (Table 3). Field pea had the highest percentage of NRA in the shoot (an

318


TA B L E 2. Total and speci®c absorption of nitrate by ®ve grain legumes grown at 0´2 (N1) and 2 mM (N2) nitrate during 0± 7, 7±14 and 14±21 d of nitrate treatment Days after nitrate addition 0±7 Species Total nitrate absorption (mg N per plant) Chickpea White lupin Narrow-leafed lupin Field pea Yellow lupin LSD (P = 0´05) Species Nitrate Species 3 nitrate Speci®c nitrate absorption rate (mg N g±1 d. wt d±1) Chickpea White lupin Narrow-leafed lupin Field pea Yellow lupin LSD (P = 0´05) Species Nitrate Species 3 nitrate

7±14

14±21

N1

N2

N1

N2

N1

N2

1´8 3´2 2´3 2´7 2´9

5´1 16´6 3´4 13´2 3´1

2´9 5´3 3´8 3´8 5´4

16´8 40´3 12´5 29´8 16´9

5´9 11´3 8´4 8´4 10´6

25´5 67´3 37´5 30´0 36´3

2´0 1´3 2´8 0´78 0´59 0´82 0´91 1´37

3´8 2´4 5´4 2´41 3´13 1´68 4´88 2´90

1´03 0´65 1´45

0´96 0´55 0´90 0´87 1´09

5´9 3´7 8´3 5´94 3´90 3´05 8´19 3´83

0´78 0´49 1´10

1´15 0´67 1´06 1´45 1´18

4´93 3´88 4´25 4´31 3´45 N.S. 0´82 N.S.

All plants were nodulated and received nitrate treatments starting from 22 d after germination. N.S., Not signi®cant at P = 0.05.

average of 96 %), followed by chickpea (89 %), yellow lupin (76 %), narrow-leafed lupin (73 %) and white lupin (57 %). Increasing nitrate supply led to an increase in the percentage of NRA in shoot, except for yellow lupin on day 7 and white lupin on day 21. Furthermore, the percentage of NRA in shoots increased in the ®rst 14 d and then decreased. Irrespective of plant species, nitrate level and sampling time, there was a positive relationship between speci®c nitrate uptake and NRA in shoots (r2 = 0´47, P < 0´001), but not in roots. Chemical composition

Prior to addition of nitrate, the nitrogen concentration in shoots was lowest in chickpea and highest in yellow lupin (Table 4). Addition of 0´2 mM nitrate increased the nitrogen concentration in chickpea, narrow-leafed lupin and ®eld pea, but reduced it in white lupin and yellow lupin in the ®rst week. Nitrogen concentrations in all species increased by day 14, and remained unchanged in chickpea and white lupin but decreased in the other species by day 21. Nitrogen concentrations were higher in plants grown at 2 mM nitrate than those in plants grown at 0´2 mM nitrate for all species except ®eld pea on day 14 and chickpea on day 21. Field pea had the highest N concentration in shoots on day 7, whereas ®eld pea and white lupin had lower N concentrations than the others on day 21. Prior to the addition of nitrate, the nitrogen concentration in roots was lowest in chickpea and highest in ®eld pea (Table 4). Species and nitrate level had less effect on N concentration in roots than in shoots.

At 0´2 mM nitrate, most plant N was derived from N2 ®xation, with the fraction for chickpea and narrow-leafed lupin being smaller than that for other species on day 14, and that for ®eld pea being smaller than that for other species on day 21. At 2 mM nitrate, the percentage plant N derived from N2 ®xation was highest in yellow lupin, followed by narrow-leafed lupin, white lupin, ®eld pea and chickpea. It decreased with time in all species (Table 4). The concentration of non-N excess cations in plants was highest in chickpea on days 0 and 7, and in narrow-leafed lupin on days 14 and 21 (Table 5). Increasing the nitrate level from 0´2 to 2 mM led to an increase in non-N excess cations in all species, with the increase being greatest in ®eld pea and smallest in narrow-leafed lupin. For individual elements (data not shown), narrow-leafed lupin had the highest concentrations of Ca, Mg and S. Chickpea and yellow lupin had higher concentrations of Ca and Mg than other species, and yellow lupin and ®eld pea had higher concentrations of S than other species. Increasing the nitrate supply generally increased the concentrations of Ca and Mg, but lowered those of S and Cl. Concentrations of K, Na and P were less affected by species and nitrate treatments than concentrations of other nutrients. H+/OH± release

At 0´2 mM nitrate, there was net H+ release in all species except chickpea in the ®rst 2 weeks (Table 6). Total proton release was highest in white lupin, followed by yellow lupin, narrow-leafed lupin and ®eld pea, and it increased


319

TA B L E 3. Nitrate reductase activity (NRA) in shoots and roots, and percentage of NRA distributed in shoots of ®ve grain legumes grown at 0´2 (N1) and 2 mM (N2) nitrate for various durations Days after nitrate addition 0

7

Species Nitrate reductase in shoot (mmol g±1 d. wt h±1) Chickpea White lupin Narrow-leafed lupin Field pea Yellow lupin LSD (P = 0´05) Species Nitrate Species 3 nitrate Nitrate reductase in root (mmol g±1 d. wt h±1) Chickpea White lupin Narrow-leafed lupin Field pea Yellow lupin LSD (P = 0´05) Species Nitrate Species 3 nitrate % NRA in shoot Chickpea White lupin Narrow-leafed Field pea Yellow lupin

0´24 0´18 0´11 0´93 0´43

14

N1

N2

N1

N2

N1

N2

3´5 0´9 1´9 6´8 2´6

12´3 3´8 4´9 21. 9 3´4

10´0 0´9 1´1 10´1 2´7

18´3 5´7 10´1 20´0 5´4

4´8 1´3 1´3 8´1 2´5

13´3 2´0 4´6 14´2 4´9

0´34

4´7 3´0 6´6

0´15 0´29 0´60 1´02 0´25

21

6´2 3´9 N.S.

0´81 1´31 1´54 0´98 2´19

2´23 4´50 3´46 1´93 5´84

0´45

1´16 1´50 1´20 1´02 1´08

1´65 1´05 N.S.

3´7 2´3 N.S. 1´20 4´80 2´38 1´09 1´38

1´23 2´47 1´32 1´63 2´72

0´90 0´57 1´27

82 61 59 94 70

88 66 69 97 59

93 57 84 96 82

2´11 8´27 2´36 1´48 2´16 N.S. N.S. N.S.

97 71 88 99 89

85 50 60 95 71

91 35 76 97 83

All plants were nodulated and received nitrate treatments starting from 22 d after germination. N.S., Not signi®cant at P = 0.05.

TA B L E 4. Nitrogen concentrations in shoots and roots, and percentage of plant nitrogen derived from N2 ®xation of ®ve grain legumes grown at 0´2 (N1) and 2 mM (N2) nitrate for various durations Days after nitrate addition 0 Species N concentration in shoot (mg g±1 d. wt) Chickpea White lupin Narrow-leafed lupin Field pea Yellow lupin N concentration in root (mg g±1 d. wt) Chickpea White lupin Narrow-leafed lupin Field pea Yellow lupin % plant N derived from N2 ®xation Chickpea White lupin Narrow-leafed lupin Field pea Yellow lupin

7

14

21

N1

N2

N1

N2

N1

N2

16´5 27´8 29´5 25´0 36´3

25´6 21´9 31´9 35´4 30´6

34´1 32´8 39´6 45´6 33´5

35´2 26´1 40´3 39´2 40´2

42´2 37´0 44´5 39´7 41´7

35´3 26´6 34´4 28´8 33´7

35´2 35´2 38´5 34´5 38´2

16´6 23´7 17´1 29´9 24´0

26´1 27´8 22´6 31´9 31´4

28´4 32´9 26´5 38´6 33´6

32´8 29´2 27´2 34´2 36´2

32´6 32´3 25´9 38´2 36´7

36´3 30´0 27´5 34´5 38´2

32´2 35´3 29´0 33´7 33´0

100 100 100 100 100

63 77 62 83 70

12 39 63 42 65

63 82 74 81 83

0 18 25 0 57

76 77 73 62 75

3 6 17 0 24

All plants were nodulated and received nitrate treatments starting from 22 d after germination. Bulked samples of three replicates were used for analysis.

320


TA B L E 5. Concentrations of non-N excess cations (mol kg±1 d. wt) in shoots and roots of ®ve grain legumes grown at 0´2 (N1) and 2 mM (N2) nitrate for various durations Days after nitrate addition 0 Species Shoot Chickpea White lupin Narrow-leafed lupin Field pea Yellow lupin Root Chickpea White lupin Narrow-leafed lupin Field pea Yellow lupin

7

14

21

N1

N2

N1

N2

N1

N2

1´45 1´20 1´46 1´01 1´20

1´55 1´14 1´62 1´28 1´48

1´76 1´56 1´83 1´80 1´47

1´98 1´49 2´28 1´52 1´92

2´29 1´90 2´46 1´93 2´16

1´72 1´23 1´86 1´26 1´47

1´97 1´68 2´19 2´07 2´10

1´53 0´83 1´31 0´76 0´99

1´19 0´90 0´99 0´72 0´81

1´86 1´38 1´51 1´24 1´13

0´86 0´97 0´82 0´71 0´49

1´73 1´63 0´96 1´27 0´60

0´94 0´82 1´01 0´84 0´43

1´70 1´77 1´18 1´08 0´62

All plants were nodulated and received nitrate treatments starting from 22 d after germination. Bulked samples of three replicates were used for analysis.

TA B L E 6. Total and speci®c net proton release by ®ve grain legumes grown at 0´2 (N1) and 2 mM (N2) nitrate during 0±7, 7±14 and 14±21 d of nitrate treatment Days after nitrate addition 0±7 Species Total proton release (mmol per plant) Chickpea White lupin Narrow-leafed lupin Field pea Yellow lupin LSD (P = 0´05) Species Nitrate Species 3 nitrate Speci®c proton release (mmol g±1 dry root d±1) Chickpea White lupin Narrow-leafed lupin Field pea Yellow lupin LSD (P = 0´05) Species 3 nitrate Species Nitrate

7±14

14±21

N1

N2

N1

N2

N1

N2

±29 294 126 57 109

±217 ±190 51 ±490 24

±1 496 158 86 207

±265 ±390 ±16 ±641 ±23

183 645 336 99 480

±111 ±401 81 ±433 138

13 9 19 ±5´9 16´5 20´7 6´4 16´7

19 12 27 ±46´8 ±10´7 8´3 ±55´5 4´1

±0´1 16´2 15´9 5´4 15´0

2´4 1´5 3´4

92 58 131 ±36´7 ±11´8 ±1´2 ±37´9 ±1´6

2´0 1´2 2´8

14´5 12´6 16´7 3´8 16´1

±7´2 ±6´8 3´4 ±18´6 4´2 1´9 1´2 2´7

All plants were nodulated and received nitrate treatments starting from 22 d after germination.

with time. Speci®c H+ release (the amount of H+ produced per unit biomass per day) was highest in narrow-leafed lupin, followed by yellow lupin, white lupin, ®eld pea, and chickpea in the ®rst 7 d of nitrate treatment. With time, the speci®c H+ release increased in chickpea and decreased slightly in white lupin, narrow-leafed lupin and ®eld pea. When 2 mM nitrate was supplied, narrow-leafed lupin and yellow lupin still showed net H+ release, whereas the other

species showed net OH± release, with ®eld pea releasing most OH± and white lupin the least (Table 6). Irrespective of legume species and nitrate level, speci®c proton release was negatively correlated with nitrate uptake and NRA in shoots (Fig. 1) but not with NRA in roots. There was a good correlation between measured amounts of proton release and proton release calculated using the equation of Troelstra et al. (1985) (Fig. 2).


321

F I G . 1. Relationships between speci®c proton release and speci®c nitrate uptake during 0±7, 7±14 and 14±21 d of treatment, and nitrate reductase activity in shoots measured on days 7, 14 and 21 of ®ve nodulated grain legumes grown at 0´2 mM (N1) and 2 mM nitrate (N2). Nitrate treatments were imposed 22 d after germination.

F I G . 2. Relationship between measured and calculated amounts of proton release (r = 0´73) by ®ve nodulated grain legumes grown at 0´2 mM (N1) and 2 mM nitrate (N2) during 0±7, 7±14 and 14±21 d of treatment. Nitrate treatments were imposed 22 d after germination. Amounts of proton release were calculated using the equation of Troelstra et al. (1985).

DIS CUS S ION Nitrate uptake

The present study has shown striking differences among species in the ability of nodulated plants to take up nitrate from culture solution. In general, ®eld pea had the greatest speci®c absorption rate, especially at 2 mM nitrate supply, followed by chickpea, yellow lupin, narrow-leafed lupin and white lupin. Differences among species in nitrate uptake were related to differences in NRA in the shoot. The high nitrate absorption ability of ®eld pea and chickpea was also

attributed to an inhibition of N2 ®xation by nitrate. For example, N2 ®xation was completely inhibited by 2 mM nitrate in chickpea and ®eld pea after 7 d exposure to nitrate. It has been shown that high nitrate concentration can inhibit nodulation in many legume species (e.g. Harper and Gibson, 1984). In the present study, all the plants were well nodulated before nitrate treatments were imposed. The inhibition of N2 ®xation by nitrate must have resulted from the effect of nitrate on nodule development, senescence and/ or function. The results also suggest that nodule development/function is more sensitive to nitrate in ®eld pea and chickpea than in Lupinus species. Whereas ®eld pea and chickpea are better adapted to high pH soils, the three lupin species are better adapted to acid soils (Jayasundara et al., 1998). In acid soils, ammonium is generally the dominant nitrogen form. Why nodule function of the Lupinus species is tolerant to nitrate is not known. Differences in nitrate uptake among species and genotypes have been reported previously for legumes grown in nutrient solution (Chalifour and Nelson, 1988; Laine et al., 1993; Dunbabin et al., 2001) and in soils (Tang et al., 1999). In a pot experiment, increasing the supply of nitrate increased the uptake of nitrate by nodulated plants, with the proportion of plant N derived from nitrate uptake ranging from 14 to 41 % among seven grain legumes supplied with nitrate at a rate of 14 mg N per kg soil, and from 57 to 78 % when supplied with nitrate at 57 mg N per kg of soil (Tang et al., 1999). Nitrate reductase activity and distribution

The species tested in the present study are legumes of temperate origin, and are generally considered to be root nitrate assimilators. However, over 50 % of total plant NRA was found in the shoots, even when plants were supplied with 0´2 mM nitrate, a concentration likely to occur in agricultural land (Anderson et al., 1998). The distribution of

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NRA in the shoots in the present study was thus much higher than values reported for chickpea, white lupin, narrowleafed lupin and ®eld pea at comparable or higher external concentrations of nitrate (Andrews, 1986; Wallace, 1986). The discrepancy between this and previous studies could be due to the effects of plant species, cultivar, nodulation and age, or may result from experimental conditions, but nodulation appeared to be a major factor. In the present study, nitrate was supplied 22 d after germination when all the plants were fully nodulated. Relative to non-nodulated plants, nodulated plants might increase NRA more in shoots than in roots in response to nitrate supply. For example, Ligero et al. (1987) showed that nodulated ®eld pea grown at 2 mM nitrate had only one-third of the root NRA, but twice the stem NRA of non-nodulated plants. It was claimed that nodules might act as a sink for photosynthates and thus diminish the energy and reducing power supplies for nitrate assimilation, especially in roots (Ligero et al., 1987). Moreover, in most previous studies, plants were grown in sand or vermiculite where total nitrate available to plants was much less than that in nutrient solution, such that they would take up less nitrate than those grown in nutrient solution at an equivalent nitrate concentration. There was a clear difference among species in NRA distribution. This difference among species was not in the principal site of NRA, but in their different responses to external nitrate supply. While chickpea and ®eld pea showed a greater increase in shoot NRA, the Lupinus species, particularly white lupin, showed a greater increase in root NRA, as external nitrate concentration increased. This led to a higher proportion of NRA in the shoots of chickpea and ®eld pea than in those of the lupin species. The results are in general agreement with previous reports for the same species, with the exception of yellow lupin (Andrews, 1986; Wallace, 1986). Similar to previous reports for many species of temperate origin, including legumes and non-legumes (Andrews, 1986), increasing the nitrate supply increased NRA more in the shoots than in the roots of the ®ve species in the present study (with the exception of white lupin on day 21). It has been suggested that the increased rate of nitrate uptake with increasing external nitrate concentration could not induce an increase in NR synthesis in the roots of temperate species (Andrews, 1986). The rate of nitrate assimilation in the roots would thus be unable to keep pace with the increased rate of nitrate uptake, such that a larger proportion of absorbed nitrate would remain unassimilated, and would be transported to, and assimilated in, the shoots. It has been shown that nitrate concentrations in xylem sap of white lupin increased as external nitrate concentration increased (Atkins et al., 1979; Andrews, 1986). In the present study, plant nitrate uptake was correlated with NRA in the shoots but not in the roots across the ®ve species. The reason for the decreased percentage of NRA in shoots of white lupin with increasing nitrate supply on day 21 is unknown. White lupin generally forms cluster roots that proliferate over time. Whether the response to external nitrate of NRA in the cluster roots differs from that in non-cluster roots deserves investigation.

Acid production

In the present study, proton release varied markedly among species and with the level of nitrate supply. At 0´2 mM nitrate supply, narrow-leafed lupin had the highest speci®c proton release, followed by yellow lupin, white lupin, ®eld pea and chickpea. This variation in proton release generally resulted from the difference in excess uptake of cations over anions, as supported by the high correlation between the measured amounts of proton release and the values calculated from the equation based on the ionic balance model (Troelstra et al., 1985). In addition, because nitrate is the dominant anion, such species variation was also re¯ected in the ability to take up nitrate, and reduced N2 ®xation. Furthermore, the deviation between the measured and calculated proton release might be due, in part, to the difference between treatments in the amount of unassimilated nitrate in plants that was not considered in the calculation. The ranking of species in terms of proton release (except for chickpea grown at 0´2 mM nitrate) is in agreement with previous studies in nutrient solution (McLay et al., 1997) and soil culture (Tang et al., 1999). However, in these previous studies involving nine species reliant on N2 ®xation, or grown at various levels of nitrate, chickpea was the species that released most protons. In contrast, in the present study, chickpea released fewer protons at 0´2 mM nitrate and more OH± at 2 mM nitrate than Lupinus species. The reason for this discrepancy between studies is not clear. One possible explanation is that nitrate impairment of nodule function in chickpea was greater in solution culture than in soil. It was evident that the percentage of plant N derived from N2 ®xation was much lower in the present study than in the previous soil study. Narrow-leafed lupin and yellow lupin did not excrete OH±, even at high nitrate supply. This phenomenon might be explained by the release of organic anions instead of OH± during nitrate uptake and assimilation, with the result that the pH of the root media did not rise (Bolan et al., 1991; Loss et al., 1994). In addition, the marked tolerance to high concentrations of nitrate of N2 ®xation in these two species also contributed to proton release. Uptake and assimilation of nitrate involve OH± release or + H in¯ux, with the amount of OH± produced varying according to whether nitrate is assimilated in the shoots or roots (Bolan et al., 1991). It was expected that release of OH± would be more related to nitrate reduction and thus to NRA in the roots than in the shoots. By contrast, the present study showed that the amounts of protons released by the ®ve legume species under different nitrate supplies were highly correlated with NRA in the shoots but not with NRA in the roots. The results suggest that nitrate assimilation in shoots is a major driving force for nitrate uptake, which, in turn, controls the cation±anion balance and consequently in¯uences H+/OH± release. In conclusion, nitrate nutrition had a strong in¯uence on proton release by nodulated grain legumes. Increasing nitrate supply reduced acid production and increased the levels of non-N excess cations in plants. The species varied greatly in H+/OH± release. This variation was related to their

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