Inhibition of Nodule Functioning in Cowpea by a Xanthine ... - NCBI

Plant Physiol. (1988) 88, 1229-1234 0032-0889/88/88/1229/06/$0 1.00/0

Inhibition of Nodule Functioning in Cowpea by a Xanthine Oxidoreductase Inhibitor, Allopurinol Received for publication May 2, 1988 and in revised form June 27, 1988

CRAIG A. ATKINS*, PAUL J. SANFORD, PAUL J. STORER, AND JOHN S. PATE Department of Botany, The University of Western Australia, Nedlands Western Australia 6009, Australia ABSTRACT Allopurinol (lH-pyrazolo-13,4-dpyrimidine-4-ol), an inhibitor of xanthine oxidation in ureide-producing nodulated legumes, was taken up from the rooting medium, translocated in xylem, and transferred to nodules of both the ureide-forming cowpea (Vigna unguiculata L. Walp.) and the amide-forming white lupin (Lupinus albus L.). Cowpea suffered severe nitrogen deficiency, extreme chlorosis, and reduced growth, whereas lupin was unaffected by the inhibitor. Similar results were obtained with oxypurinol (lH-pyrazolo-13,4-dpyrimidine-4,6-diol). Xylem composition of symbiotic cowpea was markedly changed by allopurinol. Ureides fell to a very low level, but xanthine and, to a lesser extent, hypoxanthine increased markedly. Xylem glutamine was also reduced, but there was little change in other amino acids. Nitrogenase (EC 1.7.99.2) activity of intact nodulated plants or nodulated root segments of plants treated with allopurinol or oxypurinol for 24 hours or more was severely inhibited in cowpea but unaffected in lupin for periods of exposure up to 9 days. Nitrogenase activity of slices of nodules prepared from allopurinol-treated cowpea showed inhibition comparable to that of intact plants. Breis prepared from nodules of treated plants showed no reduction in nitrogenase, nor was there reduction in activity of breis following addition of allopurinol, xanthine, or a range of purine pathway intermediates. Increasing the 02 concentration in assays above 20% (volume/volume) reversed inhibition of nitrogenase by allopurinol in intact nodulated roots. It was concluded for cowpea that allopurinol not only inhibited ureide synthesis but also caused inhibition of nitrogenase activity, thereby leading to progressive dysfunction and eventual senescence of nodules. The mechanistic relationships between inhibition of ureide biosynthesis, changes in gaseous diffusion resistance, and reduced nitrogenase activity remain obscure.

Nodulated tropical legumes, such as soybean, cowpea, and lima bean, which form ureides (allantoin and allantoic acid) as major translocated products of N2 fixation, were recently shown to exhibit severe, progressive chlorosis when exposed through the rooting medium to a xanthine oxidoreductase inhibitor, allopurinol (18). Application of combined nitrogen prevented the onset of such chlorosis, and there was apparently no inhibitory effect of allopurinol on the nodulated root systems of a range of temperate symbioses (pea, lupin, alfalfa, and clover) which form amides, rather than ureides, as products of fixation (18). Although the specificity of this inhibitory effect of allopurinol on legumes has not been clearly established, these observations suggest that, when ureide synthesis has been blocked, other solutes of N are either not formed from fixed N or are not translocated in amounts sufficient to prevent N deficiency in shoots of the host. However, since both glutamine and/or aspar-

agine are formed and exported concomitantly with ureides in tropical legumes (7, 12), it seems surprising that amide export does not increase to compensate for the lack of ureide synthesis. Indeed, there would be no apparent need for increased rates of glutamine synthetase activity, as all fixed N used in ureide formation is likely to be derived, directly or indirectly, from the amide group of glutamine (1, 13). Furthermore, in cowpea, glutamine export predominates over ureide export under conditions of Ar:02 treatment when enzymes of ureide biosynthesis have been temporarily reduced in activity relative to glutamine synthetase (6). The logical conclusion from the above is that, in addition to inhibiting ureide synthesis, allopurinol affects ureide-forming symbioses by directly or indirectly inhibiting component processes of nodule functioning which are unrelated to xanthine oxidoreductase. The present study examines more closely the uptake, translocation, and inhibitory effects of allopurinol applied to the functional nodulated root systems of ureide-forming (cowpea) and amide-forming (lupin) symbioses.

MATERIALS AND METHODS Plant Material. Cowpea (Vigna unguiculata L. Walp. cv Vita 3), effectively nodulated with Bradyrhizobium strain CB 756, and white lupin (Lupinus albus L. cv Ultra), effectively nodulated with Rhizobium strain WU 425, were grown with nutrient solution free of combined N in sand culture in a naturally lit glasshouse (2). The cowpea symbiosis was also grown in aerated liquid culture and in sand culture using the 3.5-L containers and conditions described earlier (5, 6). Exposure to Inhibitors. Allopurinol (lH-pyrazolo-[3,4-d]pyrimidine-4-ol) and oxypurinol (lH-pyrazolo-[3,4-dJpyrimidine4,6-diol) were purchased from Sigma (St. Louis, MO) and used without further purification. Stock solutions of inhibitors were prepared with 0.01 M KOH and diluted into nutrient solutions to a final concentration of 0.5 mm. The same amount of KOH was added to control nutrient solutions. The nutrient solutions of liquid cultures (with or without allopurinol) were changed every 1 to 2 d. Sand cultures were flushed through with nutrient solution (with or without allopurinol) every 2 d. Nitrogenase Assay of Intact Plants and Nodulated Roots. Nitrogenase (EC 1.7.99.2) activity of plants in both liquid and sand culture was assayed by acetylene reduction with an open, continuous flow system similar to that described by Minchin et al. (10). Maximum rates of gas exchange were recorded after 10 min and remained stable for up to 40 min whether or not inhibitor was present. Following assay, the culture vessels were well sparged with air to displace any residual ethylene and acetylene. Nitrogenase activity of plants grown in sand culture was also assayed with freshly harvested nodulated root segments as described previously (5, 6). Preparation and Nitrogenase Assay of Nodule Slices and Breis. Freshly harvested cowpea nodules (5-10 g fresh weight) were

1229

ATKINS ET AL.

1230

washed in deionized water and chopped into 0.5- to 1-mm slices in a chilled medium containing 10 mM 3-[N-morpholino]propanesulfonic acid-KOH (pH 7.5). Slices were transferred to 34mL serum vials containing 2 mL of the chopping medium together with 10 mm sodium pyruvate and thoroughly gassed with 40% 02:60% N2 (v/v) before sealing. Acetylene was added to 10% (v/v), and vials were incubated at 30°C in a shaking bath to determine nitrogenase activity. The fresh weight of tissue in each vial was determined at the end of the assay period. Where the effect ofallopurinol on nitrogenase activity of slices was being assessed with time, the tissue was incubated with the inhibitor for varying periods up to 6 h, but acetylene was added to 10% only 30 min prior to assay at each point in the time course. Breis containing intact bacteroids were prepared from cowpea nodules under anaerobic conditions by a modification of the method of Salminen (16). Freshly harvested nodules (6-10 g fresh weight) were gently crushed in a chilled mortar and pestle with 15 mL of chilled (2-3°C) breaking medium containing 80 mM Tes (pH 7.1), 2 mM Mg C12, 200 mm ascorbic acid, 5 mm DTT, and 2% (w/v) PEG 4000. Immediately prior to use, a small amount of sodium dithionite (0.2-0.5 mg- mL-') was added to the breaking medium so that there was a slight excess when tested with methyl viologen indicator paper. Samples of the brei (1 mL) were added, under a stream of Ar, to 34-mL serum vials containing 2 mL of degassed 100 mm (pH 7.4) phosphate buffer with 20 mm sodium pyruvate. The vials were thoroughly gassed with 2.5% 02:97.5% Ar (or 97.5% N2) (v/v) and sealed. After addition of 3 mL C2H2, the vials were incubated at 30C in a shaking bath, and 0.5-mL gas samples were taken at 30-min intervals for ethylene assay. Nodule breis prepared in this way showed linear rates of acetylene reduction (4-10 ,umol min' . mL-') for up to 4 h when pyruvate, malate, succinate, or 3-phosphoglyceric acid was included and a sharp optimum between 2 and 3% (v/v) 02. When an organic acid was omitted from the reaction mixture, or when 20 mm glucose or sucrose was substituted, breis showed negligible acetylene reduction at any 02 level. Gas mixtures were generated with mixing pumps, and the 02 levels of atmospheres within vials at the beginning

Plant Physiol. Vol. 88, 1988

and end of assay periods were checked by gas chromatography (6). Collection and Analysis of Xylem Exudate. Root bleeding exudate was collected for 30 to 40 min after decapitation of both cowpea and white lupin plants at varying times after the addition of allopurinol and at the same time from plants in control pots. Sap samples from five plants, each from separate pots, were pooled and stored in polypropylene containers in liquid N2 prior to analysis. Amino acids in sap samples were separated and measured by an ion exchange HPLC technique which incorporated Li buffers and postcolumn ninhydrin detection. Ureides were measured as the phenylhydrazone of glyoxylate (17) or by HPLC anion exchange chromatography (15). Purines and allopurinol were separated and assayed by the HPLC reverse phase gradient ion-pairing and ion-suppression procedures described previously (4, 7). Extraction of Nodules and Assay of Solutes. Water-soluble materials were recovered from ethanol extracts of nodules as described previously (3, 7) and assayed for purines and allopurinol as above. Harvest of Plant Material for Determination of Dry Matter and N Content. Effects of allopurinol and oxypurinol on growth and N nutrition of cowpea were assessed by harvesting whole plants from sand or water culture prior to (21 d after sowing) and 24 d (45 d after sowing) after exposure to the inhibitors. In each case, six replicate containers, each of five plants, were

0.8

I0

0

CL~~~~~~~A E

hypoxanthin

0.4

EE

Table I. Effects ofAllopurinol, Combined Nitrogen, and Oxypurinol Treatment in Sand or Liquid Culture A. Interaction of allopurinol treatment and application of combined nitrogen in nodulated cowpea grown in sand culture. Effectively nodulated plants (21 d old) were exposed to nutrient solutions containing 0 or 0.5 mM allopurinol, 0.5 mm allopurinol + 5 mm NH4NO3, or 5 mM NH4NO3. B. Effect of 0.5 mM allopurinol or 0.5 mM oxypurinol on increments of dry weight and fixed N in nodulated cowpea grown in aerated liquid culture. Increments over 21 to 45 da Treatment Dry wt Nitrogen

g-plant-'

mg-plant-'

A. Sand culture

Control 2.95 + 0.07b 88.40 + 1.89 + Allopurinol 1.82 ± 0.05 12.37 ± 0.90 + Allopurinol and NH4NO3 3.42 ± 0.16 60.45 ± 3.31 + NH4NO3 3.65 ± 0.17 99.03 ± 3.57 B. Water culture Control 1.01 ± 0.03 20.7 ± 1.1 + Allopurinol 1.01 ± 0.04 8.0 ± 0.6 + Oxypurinol 0.87 ± 0.06 12.6 ± 1.4 a Increments in dry weight and N were computed as the difference between plants at 21 and 45 d after sowing. At 21 d, plants in sand culture showed 0.75 ± 0.02 g dry weight and 25.83 ± 0.97 mg N-plant-', and in liquid culture 1.01 i± 0.04 g and 30.7 + 1.1 mg, respectively. b All values are means ± SE (n = 6).

0.2t

Z

0 +

7.

2h

7h

Id

2d

TIME

AFTER + AP

5d

7d

9d

FIG. 1. Contents of xanthine, hypoxanthine, and allopurinol (AP) in xylem exudate collected from nodulated sand-grown cowpea plants treated with 0

or

0.5

mmi allopurinol

for periods from 2 h to 9 d.

Table II. Allopurinol Content ofXylem Sap and in Extracts of Nodules from 35-d-Old White Lupin Plants Exposed to 0.5 mm Allopurinol in Sand Culture Pots were flushed daily with nutrient solution, free of combined N but containing allopurinol (AP), for 9 d and from 9 to 12 d with nutrient solution containing no allopurinol. Allopurinol concentration in Time after + AP Nodules Xylem sap

d

mLo'

nmol-g-' noduleft wt

2 3 4 5 9 12

13.9

54.3 75.0 99.8 318.2 445.3 203.3

19.6 43.7 74.0 174.1 15.4

1231

ALLOPURINOL INHIBITION

E

E

2h

7h

Sd 2d Id TIME AFTR +AP

7d

9d

FIG. 2. Contents of amino acids and ureides (allantoin + allantoic acid) in xylem exudate collected from nodulated sand-grown cowpea plants treated with 0 or 0.5 mm allopurinol (AP) for periods from 2 h to 9 d.

Table III. Acetylene Reduction by Excised, Intact Nodulated Roots of 35-d-Old White Lupin Plants Grown in Sand Culture and Exposed to Allopurinolfor up to 9 d Pots were flushed daily with nutrient solution containing 0 or 0.5 mM allopurinol (AP) Rate of C2H2 Reduction Time after + AP + 0.5 mM AP Control gmol-h.'/g-' nodulefr wt d 22.03 ± 3.40 2 21.83 ± 2.04a 14.96 ± 1.56 3 11.78 ± 1.94 19.53± 1.11 4 15.90±3.56 15.50 ± 0.88 5 13.03 ± 0.40 14.43 ± 1.77 9 12.98 ± 0.62 a Values are means ± SE (n = 3). 1a0~~~~

id' Ip sot

~~~+ tAP

1

\'

-

CONTROL

-!

d

30 t

20e

.2

i

I.5

i0 mmwffl..

.

i

woo

ooo

am TIME

of DAY

op

,

mooo

(h)

FIG. 4. Time course of inhibition of nitrogenase activity by 0.5 mM allopurinol (AP) applied to intact nodulated cowpea plants (solid lines) at 1800 h (@-O), or 1000 h (O-O), or to slices of nodules (dashed line). 0.5SmM

E

2

AP

4

t

6

TIME AFTER+ AP (d)

FIG. 3. Effect of 0.5 mm allopurinol (AP), applied in nutrient solution to the rooting medium of fully symbiotic nodulated 21-d-old cowpea

plants, grown in sand (dashed lines) or liquid (solid lines) culture, on rates of acetylene reduction. The assays used a continuous flow open gas exchange assay in which an air stream containing 10% (v/v) acetylene was passed through the culture vessels at a measured flow of 200 to 300 ml-min-'. Samples were taken at 10-min intervals. The values shown are the means (± SE) of maximum rates from three separate pots (four or five plants). harvested; after dry weight determination, whole plants were milled and samples were taken for total N assay by Kjeldahl

digestion and distillation. Xanthine Oxidoreductase Assay. Activity of NAD:xanthine oxidoreductase (EC 1.2.1.37) was assayed in desalted cell-free extracts of cowpea nodules by NAD reduction as described previously (3). RESULTS Effect of Allopurinol and Oxypurinol on Growth and N2 Fixation. Nodulated cowpea plants showed progressive chlorosis and

complete senescence of nodules after 24 d of culture in allopurinol. Fixation of N2 over the period was markedly reduced in treated plants whether grown in sand or water culture (Table I). The inhibition of fixation was substantially overcome by application of combined nitrogen (Table IA), indicating that, over the time course of the experiment at least, the inhibitory effects of allopurinol were largely restricted to nodule functioning. Oxypurinol which, like allopurinol, inhibited cowpea nodule xanthine oxidoreductase activity in vitro (complete inhibition at 10-5 mM oxypurinol compared with 10-6 mM allopurinol), also led to chlorosis in plants exposed to the inhibitor in the rooting medium and caused a marked reduction of N2 fixation (Table IB). By comparison with cowpea, white lupin showed no chlorosis and no inhibition of growth or of N2 fixed or reduction in nitrogenase level during prolonged exposure of plants to either of the inhibitors in sand culture (data not shown). Effect of Allopurinol on Xylem Transport of N Solutes. Allopurinol was readily detected in xylem exudates collected as bleeding sap from nodulated root systems of treated cowpea plants (Fig. 1), reaching levels similar to those in the nutrient solution (0.5 mM) by 5 d. Slightly lower, although still substantial, levels of allopurinol were translocated in the transpiration stream of white lupin (Table II). As indicated in Table II, flushing pots through with nutrient solution free of allopurinol for 3 d following 9 d of exposure led to a sharp decline in the level of the inhibitor in xylem exudate. Similar results were found for cowpea (data not shown) and suggest that the level of allopurinol in

ATKINS ET AL.

1232


Table IV. Acetylene Reduction by Excised Intact Nodulated Roots, Sliced Nodules, and Nodule Breis Sampled or Preparedfrom 21-d-Old Sand-Cultured Cowpea Plants 24, 48, or 72 h after Exposure of the Plants to 0 or 0.5 mM Allopurinol (AP) in the Rooting Medium

Time after

Material Assayed

[02]

+ 0.5 mM AP to Plants

iAsa

Rate of C2H2 Reduction Inhibition Cotl +AP due to AP

% h ,umol h-'.g-' noduleft wt %, v/v 12.3 ± 2.3 43 21 24 21.6 ± 2.3a 48 21 15.0 ± 0.7 7.1 ± 1.1 53 72 21 31.6 ± 1.9 8.4 ± 4.0 74 24 40 5.4 ± 0.2 3.8 ± 0.1 30 Sliced nodules 48 40 4.8±0.3 2.0±0.4 58 72 40 7.5 ± 0.6 1.3 ± 0.3 83 12 2.2 ± 0.1 2.5 ± 0.1 48 2.5 Nodule brei 17 3.4±0.1 72 4.1 ±0.1 2.5 ± a All values are means SE with n = 3 for whole nodulated roots and isolated bacteroids and n = 5 for nodule slices.

Whole nodulated roots

*-*+05 mM

AP

3 e 0~~~~~~~~~~

|

3

SE of

2

P~~~~

0~~ E

1

3 2 4 TIME (h) FIG. 5. Effect of addition of 0.5 mm allopurinol (AP) or 0

1

1

mM

xanthine on the progress of pyruvate-dependent acetylene reduction by breis prepared from nodules of 21-d-old cowpea plants. One mL of bacteroid suspension was equivalent to 0.2 to 0.3 g nodule (fresh weight).

xylem represented current uptake rather than reflecting extensive recycling to xylem in the transport channels of the plant. Consistent with inhibition of xanthine oxidoreductase in nodules by allopurinol, there was a sharp and substantial increase in xylem-borne xanthine and, to a lesser extent, hypoxanthine (Fig. 1), and a corresponding decline in ureides (Fig. 2). After 2 d, there was progressive reduction in the volume of xylem exudate collected from plants treated with allopurinol compared to controls. Although the reason for reduced bleeding under these conditions is not clear, it seems likely that the increasingly elevated levels of all xylem-borne solutes recovered after 2 d of treatment (Figs. 1 and 2) were, to some extent, a consequence of reduced exudation. Glutamine, which was the most prominent amino compound in xylem exudate of cowpea, was also sharply reduced in content by allopurinol treatment (Fig. 2). The other amino acids in xylem

exudate (Fig. 2) (which comprised Asp, Glu, Asn, Thr, Ser, Gly, Ala, Leu, Ile, Tyr, His, Met, Lys, Arg, and y-Abu) did not show a significant and consistent pattern of change with treatment. Effect of Allopurinol on Nitrogenase Activity. Progressive inhibition of nodule functioning in cowpea by allopurinol was readily detected by assaying nitrogenase activity of intact plants exposed to the compound in either sand or liquid culture (Fig. 3). Although under both sets of culture conditions acetylene reduction was depressed after 1 d, inhibition was much more marked in sand-grown plants compared to those in liquid culture, probably because of the potential for direct uptake of the inhibitor from the medium into nodules of sand-cultured plants but not into those of water-cultured plants. Both the roots and nodules of plants in sand were bathed in nutrient solution, whereas in liquid culture the crown nodulation zone, where the majority of nodules were located, was above the nutrient solution level so that entry of allopurinol to nodules would have been restricted to transfer from the host. Consistent with a lack of inhibitory effects of allopurinol on growth and development of nodulated white lupin, there was no detectable effect of the inhibitor on nitrogenase activity of the species (Table III), assayed using detached segments of nodulated root. Although acetylene reduction by intact cowpea plants was depressed by as much as 50% following 24 h exposure to allopurinol (Fig. 3), a more detailed examination of the time course showed that there was a considerable lag before the onset of inhibition. When allopurinol was applied at dusk, nitrogenase activity was unaffected for 18 h but was then sharply reduced after noon the following day (Fig. 4). The lag was largely abolished if allopurinol was applied to slices of nodules (Fig. 4). Inhibition of nitrogenase by allopurinol was compared in intact nodulated roots, slices of nodules, or nodule breis containing a suspension of bacteroids (Table IV). In each case the assay material was prepared from plants treated for 24, 48, or 72 h previously with 0 or 0.5 mm allopurinol. Although progressive inhibition of nitrogenase in nodulated roots and nodule slices (Table TV) reflected the situation found in intact plants (Fig. 3), the activity assayed in nodule breis was, by comparison, little affected by prior allopurinol treatment. After 72 h exposure to allopurinol, acetylene reduction by intact plants was reduced by 82% (Fig. 3), by 74% in nodulated root segments (Table IV), and by 83% in sliced nodules (Table IV), while breis prepared from nodules of treated plants showed only 17% inhibition (Table IV). Addition of 0.5 mm allopurinol or 1 mm xanthine to breis, prepared from nodules of plants previously treated or not treated with allopurinol, had a negligible effect on acetylene reduction (Fig. 5). Similar concentrations of a range of purine pathway intermediates (IMP, AMP, XMP, xanthosine, inosine,

ALLOPURINOL INHIBITION

1233

Table V. Nitrogenase Activity ofDetached Nodulated Root Segments ofSand-Cultured Plants Segments were darkened for 48 h or incubated in an open gas exchange system and supplied C02-free air for 48 h, or the root system was exposed for 24 h to 0.5 mm allopurinol. Assays were carried out separately with four ditfferent O2 concentrations in the assay vials. % (V/V) 02 in Nitrogenase Assay Treatment 50 30 40 20 gmol C2H2 formed - h -'.g-'fr wt nodule 21.9 ± 0.6 6.4 ± 1.2 17.3 ± 0.9 Control 19.9 ± 1.6a b 11.2 ± 1.6 After 48 h darkness 2.2 ± 0.1 11.4 ± 1.0 5.3 ± 1.7 After 48 h C02-free air 5.9 ± 2.4 22.2 ± 3.1 11.9 ± 1.3 22.3 ± 4.5 After 24 h + allopurinol a b ± = All values are means SE (n 3). Not assayed.

hypoxanthine) and NH4Cl were also found to have no inhibitory effect on nitrogenase activity of breis (data not shown). Increasing the external 02 tension in assays of nodulated roots taken from plants 24 h after treatment with allopurinol completely reversed the inhibition of nitrogenase (Table V). A response similar to that with allopurinol treatment was shown by nodulated roots from plants in which photosynthate supply was reduced by continuous exposure of shoots to darkness or to C02free air (Table V).

DISCUSSION The inhibitory effects of allopurinol and oxypurinol on growth and N2 fixation of cowpea (Table I), but not lupin, confirmed the qualitative observations of Triplett (18) and indicated that the allopurinol-induced chlorosis in ureide-forming symbioses was associated with severe N deficiency. Furthermore, in cowpea (Fig. 3), but not lupin (Table III), nitrogenase activity was markedly inhibited by allopurinol, suggesting that the N deficiency was a consequence of progressive nodule dysfunction. Allopurinol was taken up readily by both symbioses from the rooting medium, transported in xylem (Table II; Fig. 1) to the shoot, and detected at significant levels in extracts of nodule tissue (Table II) (7). It thus seemed unlikely that the difference in sensitivity of the two symbioses to allopurinol was a consequence of differences in uptake or transfer of the inhibitor to plant organs, including nodules. A previous study with cowpea (7) showed that inhibition of nodule xanthine oxidoreductase by allopurinol completely blocked the major flow of fixed N in the nodule to ureide export. As in the amide-forming symbiosis (lupin), neither the flow of fixed N to the root nor nitrogenase was affected by allopurinol. The most obvious explanation for the inhibition of nitrogenase activity in cowpea was a blockage of ureide synthesis in nodules. Breis prepared from nodules of allopurinol-treated cowpea plants (Table IV) showed similar rates of acetylene reduction to those from controls, indicating that inhibition in the intact nodule was not due to a reduced level of nitrogenase or to some impairment of bacteroid metabolism. Furthermore, direct exposure of isolated bacteroids (whether from treated or untreated plants) to allopurinol did not cause inhibition (Fig. 4). These results seemed to preclude a direct effect of the inhibitor on bacteroid metabolism. Similarly, an indirect inhibitory effect on nitrogenase due to accumulation of xanthine or other products of N2 fixation seemed unlikely, as their addition in relatively high concentrations to breis (Fig. 4) was without effect. Intermediates of de novo purine synthesis (i.e. from phosphoribosylpyrophosphate to IMP) were not tested, but significant accumulation of these compounds has not been detected in nodules treated with allopurinol (7). The time course for allopurinol-induced increases in xylem transport of xanthine (Fig. 1) showed that by 2 h after exposure there was significant inhibition of nodule xanthine oxidoreduc-

tase. A previous study (7) established that by 1 h after application of allopurinol to the root system, xanthine oxidation and ureide synthesis in nodules were reduced, and by 4 h were completely inhibited. In that study (7), as in the present one (Fig. 4), the onset of inhibition of acetylene reduction occurred much later, suggesting that the effect observed on nitrogenase of intact nodules had required time for the accumulation of high levels of allopurinol, and in this way it was distinct from the effect on xanthine oxidoreductase. The fact that the lag in inhibition of nitrogenase was essentially eliminated by applying allopurinol to slices of nodules (Fig. 4) was consistent with this conclusion. The results with slices of nodules would also seem to eliminate the possibility that the apparently specific effect of allopurinol on nitrogenase activity of intact cowpea plants was a consequence of some direct effect or indirect consequence (e.g. translocation of xanthine [7]) of its application on shoot metabolism. The finding that increasing 02 levels surrounding nodules effectively reversed inhibition of nitrogenase by allopurinol (Table V) suggested that one effect of the inhibitor was to cause a reduction in 02 supply, which in turn would have led to a lower rate of respiration supporting bacteroid functioning. Prolonged 02 limitation might be expected to cause progressive loss of nitrogenase and eventually senescence of nodules. Reversible adjustment of gaseous diffusion resistance in nodules has been recognized as a means to control endogenous 02 levels (9, 10, 19). The response to increased 02, following treatments expected to reduce photosynthate supply (CO2-free air, darkness; Table V), suggested that such a mechanism was a property of the cowpea symbiosis used in this study. Changes in the size of air spaces between cells of the inner cortex or of the infected medulla of the nodule have been suggested to be the principal means of diffusional resistance adjustment (20), so that disrupting the organ's diffusional barriers (by slicing nodules or crushing the plant cells as in breis), should reduce allopurinol inhibition of nitrogenase. While this was the case for breis, it was not so for nodule slices, which showed the same relative inhibition as when intact nodulated roots (Table IV) or intact plants (Fig. 3) exposed to allopurinol were assayed. Thus, both 02 limitation and reduced nitrogenase were apparently consequences of an effect of the inhibitor in the plant cell cytosol surrounding bacteroids and did not depend on complete structural integrity of the nodule. The results of this study clearly indicated that the effect of allopurinol on nodules was not restricted to inhibition of xanthine oxidoreductase. Although the possibility of a mechanistic relationship between the effect on xanthine oxidation and inhibition of nitrogen fixation in symbioses which form ureides was not eliminated, any possible connection between the two was concluded to be rather indirect. In addition to their acting as inhibitors of xanthine oxidation, both allopurinol and its hydroxylation product (1 1) oxypurinol have been suggested to have two other, quite different effects in animal tissues. Each has been shown to scavenge free radicals

1234

ATKINS ET AL.

formed in cellular metabolism (8), while allopurinol may also redox reagent transferring electrons from ferrous Fe to ferric Cyt c (14). In the absence of any information about the formation or function of free radicals of oxygen in cowpea nodules, it is not possible to assess how such reactions involving allopurinol might be related to its effect on nitrogenase. While it is conceivable that redox activity by allopurinol in vivo might interfere with normal oxidative phosphorylation leading to alterations in respiration and adjustment of gaseous diffusive resistance, it is difficult to see how this scenario could be specific to respiration of nodule tissue and restricted to ureide-forming plants. serve as a

LITERATURE CITED 1

2

3 4

5 6

ATKINS CA, L BEEVERS 1988 Synthesis, transport and utilization of translocated solutes of nitrogen. In YP Abrol, ed, Nitrogen in Higher Plants-Recent Advances/Future Outlook. John Wiley, London (in press) ATKINS CA, JS PATE, GJ GRIFFITHS, ST WHITE 1980 Economy of carbon and nitrogen in nodulated and non-nodulated (NO3-grown) cowpea (Vigna unguiculata [L.] Walp.). Plant Physiol 66: 978-983 ATKINS CA, RM RAINBIRD, JS PATE 1980 Evidence for a purine pathway of ureide synthesis in N2-fixing nodules of cowpea (Vigna unguiculata [L.] Walp.). Z Pflanzenphysiol 97: 249-260 ATKINS CA, A RITCHIE, PB ROWE, E MCCAIRNS, D SAUER 1982 De novo purine synthesis in nitrogen-fixing nodules of cowpea (Vigna unguiculata [L.] Walp.) and soybean (Glycine max L. Merr.). Plant Physiol 70: 55-60 ATKINS CA, BJ SHELP, J Kuo, MB PEOPLES, JS PATE 1984 Nitrogen nutrition and the development and senescence of nodules on cowpea seedlings. Planta 162: 316-326 ATKINS CA, BJ SHELP, PJ STORER, JS PATE 1984 Nitrogen nutrition and the development of biochemical functions associated with nitrogen fixation and ammonia assimilation of nodules on cowpea seedlings. Planta 162: 327-333


7 ATKINS CA, PJ STORER, JS PATE 1988 Pathways of nitrogen assimilation in cowpea nodules studied using "5N2 and allopurinol. Plant Physiol 86: 204207 8 DAs DK, RM ENGELMAN, R CLEMENT, H OTANI, MR PRASAD, PS RAo 1987 Role of xanthine-oxidase inhibitor as free radical scavenger: a novel mechanism ofaction of allopurinol and oxypurinol in myocardial salvage. Biochem Biophys Res Commun 148: 314-3 19 9 MINCHIN FR, JE SHEEHY, IM MINGUEZ, JF WITTY 1985 Characterization of the resistance to oxygen diffusion in legume nodules. Ann Bot 55: 53-60 10 MINCHIN FR, JF WITTY, JE SHEEHY, M MULLER 1983 A major error in the acetylene reduction assay; decreases in nodular activity under assay conditions. J Expt Bot 34: 641-649 11 Ni FHAOLAN I, MP COUGHLAN 1978 Effects of allopurinol and of oxypurinol on turkey liver xanthine dehydrogenase. FEBS Lett 90: 305-308 12 OHYAMA T, K KUMAZAWA 1978 Incorporation of '5N into various nitrogenous compounds in intact soybean nodules after exposure to "N2 gas. Soil Sci Plant Nutr 24: 525-533 13 PATE JS, CA ATKINS 1983 Nitrogen uptake, transport and utilization in nitrogen fixation. In WJ Broughton, ed. Ecology of Nitrogen Fixation, vol 1. Clarendon Press, Oxford, pp 245-298 14 PETERSON DA, B KELLY, JM GERRARD 1986 Allopurinol can act as an electron transfer agent. Is this relevant during reperfusion injury? Biochem Biophys Res Commun 137: 76-79 15 RAINBIRD RM, JH THORNE, RWF HARDY 1984 Role of amides, amino acids and ureides in the nutrition of developing soybean seeds. Plant Physiol 74: 329-334 16 SALMINEN SO 1981 Effect of NH4' on nitrogenase activity in nodule breis and bacteroids from Pisum sativum L. Biochim Biophys Acta 658: 1-9 17 TRIJBELS F, GD VOGELS 1966 Degradation of allantoin by Pseudomonas acidovorans. Biochim Biophys Acta 113: 292-301 18 TRIPLE-r EW 1986 Two indirect methods for detecting ureide synthesis by

nodulated legumes. Plant Physiol 81: 566-571 19 WiTTY JF, FR MINCHIN, JE SHEEHY, IM MINGUEZ 1984 Acetylene induced changes in the oxygen diffusion resistance and nitrogenase activity oflegume root nodules. Ann Bot 53: 13-20 20 WITTY JF, L SC0T, NP REUSBECH 1987 Direct evidence for changes in the resistance of legume root nodules to 02 diffusion. J Expt Bot 38: 1129-1140