Proline Fed to Intact Soybean Plants Influences Acetylene ... - NCBI

Received for publication June 18, 1991 Accepted October 30, 1991

Plant Physiol. (1992) 98, 1020-1028 0032-0889/92/98/1 020/09/$01 .00/0

Proline Fed to Intact Soybean Plants Influences Acetylene Reducing Activity and Content and Metabolism of Proline in Bacteroids' Yuxian Zhu, Georgia Shearer, and Daniel H. Kohl* Biology Department, Washington University, St. Louis, Missouri 63130 oids have dicarboxylate transport systems (30), a desirable property for an energy-supplying metabolite. Among the evidence that dicarboxylic acids can support N2 fixation is the

ABSTRACT

Supplying L-proline to the root system of intact soybean (Glycine max [L.] Merr.) plants stimulated acetylene reducing activity to the same extent as did supplying succinate. Feeding L-proline also caused an increase in bacteroid proline dehydrogenase activity that was highly correlated with the increase in acetylenereducing activity. Twenty-four hours after irrigating with L-proline, endogenous proline content had increased in host cell cytoplasm and bacteroids, about three- and eightfold, respectively. In bacteroids, proline concentration was calculated to be at least 3.5 millimolar. In experiments in which [U-14C]L-proline was supplied to uprooted, intact plants incubated in aerated solution, 14Clabeled products of proline metabolism, as well as [14CJproline itself, accumulated in both host cells and bacteroids. When plants were incubated in aerated solutions containing [5-3H]L-proline, 3H-labeled proline was found in host cells and bacteroids. [3H] Pyrroline-5-carboxylate was found in bacteroids, but not host cells, after a 2-hour incubation in [5-3H]L-proline. When [U-14C]Lproline was supplied for 24 hours, a significant amount of [14C] pyrroline-5-carboxylate was found in the host cells, in contrast with the results from the shorter incubation in [5-3H]proline, although the amount in the host cells was only about half the quantity found in the bacteroids. Taken as a whole, these results indicate that proline crosses both plant and bacterial membranes under the In vivo experimental conditions utilized and are consistent with a significant role for proline as an energy source in support of bacteroid functioning. In spite of the increase in acetylene-reducing activity when proline was supplied to the root system of intact plants, proline application did not rescue stemgirdled plants from loss of acetylene-reducing activity, although succinate application did. This suggests a nonphloem route for succinate, but not proline, from roots to nodules.

Fixation of atmospheric dinitrogen is

an

report of transformation of wild-type Bradyrhizobium japonicum with a recombinant plasmid encoding Rhizobium meliloti sequences for dicarboxylic acid transport (7). Chimeric, free-living bacteria exhibited enhanced rates of succinate uptake and nitrogenase activity. These results, however, are silent on the possibility that other classes of compounds may also support either free-living bacterial or bacteroid metabolism. Reports of Fix- phenotypes formed by dicarboxylic acid transporter minus mutants (2, 12, 26) are also cited in support of a role for Krebs cycle intermediates as the energy source that fuels N2 fixation (34). Although the microsymbiont is able to proliferate, nodules infected with such mutants do not form leghemoglobin. Leghemoglobin, as well as an energy source to the bacteroid, is required for an effective symbiosis (1). Therefore, the failure of the nodules to fix N2 may have resulted from the lack of leghemoglobin rather than lack of energy source. In addition, in flow chamber experiments at physiologically realistic concentrations of free dissolved 02, the rate of N2 fixation by rigorously anaerobically isolated bacteroids decreased instantly and markedly when succinate, and to a lesser degree when malate, was added to the medium. The rate of N2 fixation increased sharply when the added dicarboxylic acid was diluted out of the medium (5). The overall efficiency of N2 fixation (NH3 produced/02 consumed) was less in the presence of either succinate or malate than it was with endogenous substrates. Further work from the same laboratory (6) established that storage in poly-0hydroxybutyrate (to the point that this polymer constituted 50-70% of dry bacteroid mass) was an important fate of exogenous succinate and malate. These authors proposed that this polymer is mobilized in support of N2 fixation when more readily available endogenous substrates are not available. Other reduced carbon compounds have been proposed as energy substrates for bacteroid N2 fixation. Herrada et al. (18) reported that, in nodules of French beans (Phaseolus vulgaris), glucose was transported across the peribacteroid membrane (the membrane of plant origin that surrounds a bacteroid or a group of bacteroids) and into naked bacteroids at rates that were the same order of magnitude as for succinate. From this, they concluded that "glucose could act as an energy-yielding substrate in functioning nodules." However, Bergersen and Tumer (5) reported that glucose was utilized only slightly by bacteroids from soybean nodules.

energy-intensive

process, requiring a total of 25 to 30 ATP/N2 fixed. As much as 10 to 30% of the total photosynthate may be used to support this process (28). In N2-fixing legumes, this energy

demand is satisfied by reduced C molecules supplied by plant cells to microsymbionts (bacteroids), which, in turn, export fixed N2, as ammonia(um), to infected host cells. The most often mentioned candidates for the compounds supplied to the bacteroids are Krebs cycle intermediates. Soybean bacter'This research was supported by grant GM38786 from the National Institutes of Health to D.H.K.

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PROLINE FED TO INTACT SOYBEANS AFFECTS BACTEROID METABOLISM

On theoretical grounds, Kahn et al. (19) postulated that fixed nitrogen, in the form of glutamate, can be used to carry reduced carbon into the bacteroid. Salminen and Streeter (27) examined the uptake of '4C-labeled metabolites and respiratory "'C production by isolated bacteroids and concluded that both dicarboxylates and glutamate could function as respiratory substrates in soybean bacteroids. Bergersen and Turner (4) provided strong support for the hypothesis that glutamate can contribute to supplying the reduced C skeletons needed for energy production linked to N2 fixation. At physiological free dissolved [02], 10 mm glutamate was used as efficiently in direct support of N2 fixation as were endogenous substrates. In addition, the kinetics of 02 consumption by bacteroids were similar for endogenous substrates and 10 mm glutamate (4). Fitzmaurice and O'Gara (13) found that R. meliloti mutants defective in their ability to utilize glutamate formed nodules with lowered nitrogenase activity, suggesting that glutamate plays an important role in the symbiosis. However, a lesion in glutamate metabolism might have widespread consequences unrelated to cellular energetics. Moreover, when intact peribacteroid units (groups of bacteroids surrounded by peribacteroid membranes) were incubated in glutamate, the rate of entry of glutamate into bacteroids was very slow compared with that for succinate (10, 18, 31), reports that Bergersen and Turner (5) take to be a very serious objection to any proposed role for glutamate. A possible role for proline as a source of energy to the bacteroid has also been proposed (20). The activity of P5CR,2 the enzyme catalyzing formation of proline from P5C, was reported to be much higher in soybean nodules than in most other plant and animal tissues (20). The activity of ProDH in soybean nodule bacteroids was comparable to that in rat liver mitochondria. In isolated organelles from soybean nodules, little ProDH activity was detected in the mitochondria, its usual locus in plant cells (29 and references therein). Based on animal models (23), it was proposed that the high P5CR activity in nodules might function to increase the activity of the oxidative limb of the pentose phosphate pathway by producing NADP+, whereas the ProDH activity (and possible subsequent dehydrogenations) in the bacteroid might supply part of the energy needed for N2 fixation (20) or other energyrequiring bacteroid processes. An additional role for the high P5CR activity might be to allow nodules to produce proline during periods of stress. Given the sensitivity of N2 fixation to water stress, this potential would provide selective advantage if proline accumulation were to play a role in protecting nodules from drought or in facilitating recovery. Another line of evidence supporting a role for proline in N2 fixation is that, in Klebsiella, the expression of the glnA (the structural gene for glutamine synthetase), nif (N2 fixation), and put (proline utilization) operons are all regulated by the ntr (nitrogen regulatory) system (22). However, the significance of proline as a compound supplying energy to the bacteroid was called into question by the results of Day et al. (9), who found that proline was not taken up at a rapid rate by isolated peribacteroid units. These investigators concluded that the peribacteroid membrane lacks a carrier(s) for 2 Abbreviations: P5C(R), pyrroline-5-carboxylate (reductase); ProDH, proline dehydrogenase; ARA, acetylene reducing activity.

1021

proline and that the observed slow uptake was via passive diffusion. In this paper, we report that exogenous proline, as well as succinate and glutamate, stimulates ARA of nodules from intact soybean plants. We also provide evidence that exogenous proline was taken up from the surrounding medium and metabolized by bacteroids in nodules of intact soybean plants. Thus, proline must have crossed the peribacteroid membrane at rates fast enough to influence metabolic events in these experiments with intact plants, despite the slow uptake of proline (compared with malate and succinate uptake) into isolated peribacteroid units. MATERIALS AND METHODS

Chemicals and Plant Material [5-3H]L-Proline (15.0 Ci/mmol) was obtained from New England Nuclear (Wilmington, DE). Nuclear magnetic resonance analysis of radioactive proline produced by this company showed that the tritium was about equally distributed between the two H atoms attached to the C-5 position of proline. [U-"'C]Proline (250 mCi/mmol) was purchased from ICN Biochemicals Inc. (Irvine, CA). Other nonradioactive chemicals were purchased from Sigma. Dowex-50W (hydrogen form, 8% cross-linked, 200-400 mesh) was purchased from Bio-Rad Laboratories. Soybean plants (Glycine max [L.] Merr. cv Williams 82) were grown in the greenhouse as previously described (21). Briefly, seeds were inoculated with Bradyrhizobium japonicum (strain 61A89, also known as USDA 110), a gift from the Nitragin Division, Lipha Chemical, Milwaukee, WI. Plants (three per pot) were grown in 20cm diameter pots filled with perlite and fertilized with N-free nutrients, except as indicated. Supplemental light (800 ,E at plant height; 14 h day length) was supplied by 1000 W Sylvania metal halide lamps. Acetylene Reduction, ProDH Activity, and Radioisotopic Assays for Proline Uptake and Metabolism The ARA assay was done with intact plants in 1-L closed chambers containing three separate plants, with triplicate chambers for each treatment. After injecting 50 mL acetylene, the chambers were incubated for 10 min prior to withdrawing three gas aliquots (0.5 mL each) for gas chromatographic analysis. Ethylene production was monitored essentially as previously described (16) with a Varian series 3700 gas chromatograph (Varian Associates, Inc., Sunnyvale, CA) fitted with a Porapak-N column. ProDH activity was assayed essentially as described earlier (15) with modifications documented in Kohl et al. (21), except that the correction factor of two (used in ref. 21) was not applied. In this assay, 3H loss from [5-3H]proline to the medium is measured. The correction factor was used previously because only one of the two H atoms is lost to water when proline is dehydrogenated. However, we have recently found that essentially all of the P5C produced is further oxidized to glutamate, resulting in the loss of both H atoms originally present on the C-5 position of proline. For this reason, we have discontinued the practice of doubling the rate calculated from tritium loss. No correction was made for any

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ZHU ET AL.

tritium isotope effect that may be associated with dehydrogenation, although our preliminary investigation (data not shown) indicates it may be very large. Preparation of Nodule Extracts

Nodules were picked, rinsed with tap water, blotted dry, weighed, then placed in a glove bag (model S-20-20, Instruments for Research and Industry, Cheltenham, PA) under N2 flow. The nodules were gently crushed with a chilled mortar and pestle with grinding buffer (2 mL/g fresh weight). A grinding buffer with high osmotic potential (100 mM Tricine, pH 8.0, 400 mm sucrose, and 2.5 mM MgCl2) was used to minimize leaching of metabolites from bacteroids. The homogenate was flushed with N2 and filtered through Miracloth (Calbiochem, La Jolla, CA). The filtrate was centrifuged at 500g for 5 min to remove cell debris. This supernatant fluid was centrifuged at 12,000g for 15 min. The supernatant fraction from the second centrifugation contained host cytosol, small organelles, and membranes and is referred to as the host cytoplasm or cytoplasmic fraction. The pellet (bacteroid fraction) contained starch granules and large organelles of host origin as well as bacteroids. This pellet was washed once, resuspended into the same volume with grinding buffer, and used to assay ProDH activity and proline content of the bacteroids. It was previously shown (20) that contaminating organelles in the bacteroid fraction did not contain enough ProDH activity to influence the measurement. To assess the level of mitochondrial proline after external proline feeding, the 5OOg supernatant was centrifuged at 3,500g for 15 min, the pellet removed, and soluble fraction spun again at 12,000g for another 15 min. Mitochondria are known not to sediment in significant number at 3500g (1 1). Both 13-hydroxybutyrate dehydrogenase activity (a marker for bacteroids) and proline content were examined in these two separate pellets. Of the total activity, 93 ± 3% and 86 ± 2% of the ,B-hydroxybutyrate dehydrogenase and proline, respectively, were found in the 3500g pellet. The mitochondria-rich 12,000g pellet with bacteroids largely removed (as judged by the ,B-hydroxybutyrate dehydrogenase activity) contained less than 15% of the proline. From this, we concluded that proline detected in the bacteroid fraction was mainly located inside bacteroids. Preparations were kept anaerobic until analysis. Preparation of Samples for Separation and Quantification of Proline Samples of bacteroids and host cytoplasm were made from 2 g fresh weight nodules. Bacteroid metabolism was stopped by incubating with 0.417 N HCI at 70°C for 5 min. After incubation, samples were centrifuged at 12,000g for 3 min. Pellets were extracted by incubating with 70% ethanol for 5 min at 70°C. After chilling on ice for 5 min, insoluble material was removed by centrifugation at 12,000g for 3 min. The insoluble material was washed once with 70% ethanol, chilled, and spun as above. The three supernatants were combined, dried in a vacuum centrifuge, and redissolved in 200 ,uL of either 1 N HCI or 0.2 N sodium bicarbonate buffer, pH 9.0, as described below. High mol wt material was removed from host cytoplasm by incubating with ethanol as described for

Plant Physiol. Vol. 98, 1992

bacteroids. The ethanol-insoluble material was washed and the two supernatants were combined, dried, and redissolved. Recovery of proline (about 75%) from either cytoplasm or bacteroid fractions was monitored in separate experiments by adding a known amount of ['4C]proline to the two fractions at the beginning of the extraction and counting before subjecting the sample to HPLC. No factor was applied to correct for partial recovery. Proline Purification and Quantification

Proline was separated from other metabolites by Dowex chromatography or HPLC. The quantity of proline taken up into fractionated components (bacteroids or host cell cytoplasm) was calculated as (cpmf/cpmm,o) x [pro]m,, where cpmf and cpmm,o are the counts/min in the biological fraction of interest and in the medium bathing the plant roots at time zero, respectively, and [pro]m,o is the concentration of proline in the medium bathing the roots at time zero. Knowing the fresh weight of nodules from which the assayed material was prepared allows us to display the results in units of nmol proline/g fresh weight.

Separation on Dowex The method of Phang et al. (24) was used, except that the elution scheme was modified. After dissolving the sample, prepared as described above, in 1 N HCI, and derivatizing any P5C in the sample with o-amino benzaldehyde, a 200-,gL sample was placed on a l-mL bed volume Dowex 50 HI column and eluted with 4.8 mL 1 N HCI, then 1 mL 1 N HCI, and 3 mL 2 N HC1. The first 5 mL included organic and acidic amino acids, the next 4 mL contained proline. After washing the column with 8 mL of 2 M HCI, the P5C- o-amino benzaldehyde adduct elutes with an additional 2 mL of 2 N NaOH. HPLC

The HPLC system (Beckman) included two model 110 A pumps, a model 420 system controller, and a sample injector. Samples, prepared as described above, were dissolved in 200 ,uL of 0.2 M sodium bicarbonate buffer (pH 9.0) and derivatized (8) with 200 ,uL dimethylaminoazobenzene sulfonyl chloride (4 nmol/mL in acetone). Samples (15-40 ,uL) were loaded onto an analytical C18 HPLC column (S5, ODS 2, phase separation, Deeside Ind. Est., Queensferry, UK) and eluted with a gradient composed of 45 mm acetate buffer (pH 4.13) and acetonitrile at 1 mL/min. The gradient used was as follows: starting and holding at 20% acetonitrile for 5 min; 20 to 70% acetonitrile over 25 min; 75 to 100% acetonitrile over 2 min; and holding at 100% acetonitrile for 5 min before returning to and reequilibrating at 20% acetonitrile (modified from ref. 8). Proline and other amino acid derivatives were detected with a model 440 detector (fixed wave length at 436 nm). Our identification and quantification of proline content was verified by amino acid analysis at the Washington University Protein Chemistry Laboratory (Beckman model 6300 with System Gold data collection, using ion-exchange chromatography, sulfonic acid functional group, with simultane-

PROLINE FED TO INTACT SOYBEANS AFFECTS BACTEROID METABOLISM ous pH, temperature, and Na-citrate gradients and detection after ninhydrin derivatization). The control and experimental samples from bacteroid fractions were eluted from 1 mL bed volume Dowex-50 columns as described above. The prolinecontaining fractions were divided, with a portion of each analyzed by HPLC, and another portion submitted to the Protein Chemistry Laboratory for analysis. There was no significant difference in results of the two methods.

RESULTS AND DISCUSSION Effects of Succinate, Glutamate, and Proline on ARA and ProDH Activity

The data in Table I show that irrigating intact, undisturbed soybean plants growing in the greenhouse in N-free medium with 20 mM proline, glutamate, or succinate solutions 24 and 12 h prior to measurement resulted in a significant stimulation of ARA: 136 ± 7, 125 ± 6, and 136 ± 7% of control values, respectively (P < 0.001 for succinate and proline and very close to 0.001 for glutamate). These enhancements are similar to those resulting from malate feeding reported by Heckmann et al. (17). Our results are consistent with all of the tested metabolites entering bacteroids, being dehydrogenated there, and, in this process, providing energy to support N2 fixation. If this interpretation is correct, then proline, as well as the other metabolites, must have crossed the peribacteroid membrane in transit from host cytoplasm to bacteroid at a sufficiently rapid rate to have a substantial physiological impact. Both glutamate- and proline-feeding significantly stimulated ProDH activity in bacteroids (P < 0.01 and 0.001, respectively) to 142 ± 14 and 197 ± 22% of control values, respectively. This latter result is consistent with an increase in either the specific activity or quantity of ProDH in response to an increase in proline content of bacteroids (vide infra). The increase in ProDH activity in response to glutamate feeding might result from accumulation of glutamate in the host cytoplasm, leading to an increase in the rate of synthesis of proline via P5C, followed by the same set of events that occur in response to proline accumulation. Succinate feeding had no effect (98 ± 10% of control) on ProDH activity. ARA increased rapidly and linearly (r = 0.98) for at least h to 133 ± 8% of the value at t = 0 in response to proline

feeding (Fig. lA). The ARA seen after 1 h (Fig. lA) is apparently sustained, judging by a somewhat higher rate measured in the longer-term experiment (Table I). ProDH activity of bacteroids also increased linearly (r = 0.99) for the first hour to 126 ± 14% of its value at t = 0 (Fig. lB). ProDH reached still higher values in the longer-term experiment; namely, 197 ± 22% of the control value (Table I). The increase in ProDH activity and ARA were highly correlated (r = 0.98, P < 0.025) during the first hour after proline feeding, a result consistent with proline dehydrogenation supplying energy for N2 fixation. The experiments for which results are reported in Table II were designed to determine whether substrates that might supply energy to support N2 fixation could relieve the impact of severe stress such as stem girdling. We have previously found that stem-girdled plants fed 50 mM succinate retained their ability to reduce acetylene (36). We were interested in knowing whether other potential energy substrates shared this trait. When 35-d-old soybean plants were girdled at the base of the stem, their ARA decreased to 17% of the control level after 2 h. This decline was not observed when 20 mm succinate was supplied immediately after stem girdling (Table II). When 20 mM L-proline solution was supplied to the stem-girdled plants, the ARA decreased almost to the same extent as the girdled plus water only plants. The ARA of 20 mm glutamatetreated plants was also significantly lower than that of control plants. The ability of succinate to sustain ARA in stem-girdled plants is consistent with succinate being able to supply the energy needed for nitrogenase activity that is denied to the nodule by stem girdling. This result also requires a path for water other than via phloem from outside the plant to infected cells within nodules. Clearly, this path can supply physiologically significant quantities of succinate under the conditions of this experiment, even if the main supply of water, and hence nutrients, to nodules is via phloem (25, 32, 33). Given the ability of exogenous proline to stimulate ARA to the same extent as does succinate in plants that were not stem girdled, it is surprising that proline had so much less effect than did succinate on sustaining ARA of stem-girdled plants. This result suggests that proline does not share with succinate nonphloem pathways to the interior of nodules. The impact of exogenous proline on both ARA and ProDH activity (Table

Table I. Effects of Exogenous Succinate, L-Glutamate, and L-Proline on Intact 35-d-old Soybean Nodule ARA These compounds (20 mm, 500 mL/20 cm diameter pot) were applied to the root system 24 h before harvesting. An additional 500 mL/pot was supplied 12 h later. Control plants received the same volume of water alone at the same times. Error terms are ± 1 SE of six replicates for ARA and three replicates for ProDH activity measurements. Treatment

ARA

of Control

LSD(o.ol) LSD(o.ool)

325 ± 17 443 ± 14 406 ± 14 442 ± 15 60.8 82.0

ProDH Activity

g-' fresh wt (±sE) 22.6 ± 2.0 22.0 ± 1.3 32.1 ± 1.2

of Control

nmol min-'

nmol min-' g9- fresh wt (±sE)

Control Succinate L-Glutamate L-Proline

1 023

100 136 ± 7 125 ± 6 136 ± 7

44.4 ± 3.2 9.8

14.8

100 98 ± 10 142 ± 14 197 ± 22


ZHU ET AL.

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Table II. Effects of Exogenous Succinate, L-Glutamate, and L-Proline on Stem-Girdled 35-d-old Soybean ARA Solutions of these compounds (20 mm, 500 mL/20 cm diameter pot) or water were applied to the root systems immediately after stem-girdling. Measurements were performed on plants harvested 2 h later. Error terms are ± 1 SE of three replicates.

0

IsE &

'5 .5a A. B. C. D. E.

;C

336 ± 33 57 ± 2 386 ± 28 119 ± 29 78 ± 5

Control Stem-girdled plus H20 only Stem-girdled plus succinate Stem-girdled plus glutamate Stem-girdled plus proline

74 105 152

LSD(o.05) LSD(o.ol) LSD(o.001)

*;> .a

*c

ARA nmol min-' g-' fresh wt (±sE)

Treatment

I

c

E as

*1aL

.5O.b

B

0

12

24

36 time -

48

60

min

Figure 1. ARA of 35-d-old soybean plants (A) and ProDH activity (B) L-proline feeding. Soybean plants were supplied with 20 mM L-proline solution (500 mL/20 cm diameter pot) for various times before harvesting. Error terms are + 1 SE of three replicates. as a function of time after

the total nodule volume (Tables 5 and 7 of ref. 3), free proline concentrations in the bacteroid would be about 0.45, 3, and 3.6 mm, even if all of the bacteroid volume were occupied by water. Because a significant fraction of the volume of bacteroids in soybean nodules is occupied by poly-f3-hydroxybutyrate particles (35), these proline concentrations are underestimates. The host cytoplasm of proline-fed nodules accumulated proline at the same rate as did the bacteroids for the first hour (Fig. 2). The rate of accumulation in the host cytoplasm fell to a very low value during the second and subsequent hours, a result consistent with the establishment of a steady-state in which the rate of export of proline from the cytoplasm to the bacteroids was equal to the rate of import of exogenous proline into host cell cytoplasm.

I) and additional results reported below strongly support the conclusion that proline can cross both root and peribacteroid membranes in intact soybeans. Apparently, however, it cannot move from outside the roots into bacteroids in the absence of phloem. Further work on conditions for proline transport across plant membranes is warranted.

800

600

-

400

-

Io

Proline Concentration in Soybean Nodules

Figure 2 shows the time course of proline accumulation (compared with its control value at t = 0) in the experiments in which the plants were fed 20 mm proline at time zero and 12 h later. As soon as 2 h after feeding proline, bacteroid proline contents had increased 6.6-fold and host cytoplasm proline content had increased about 2.5-fold. By 24 h after feeding proline, bacteroid and host cytoplasmic proline content had increased about eight- and threefold, respectively. This would seem to require transport of proline across the peribacteroid membrane at a rate sufficient to result in proline accumulation in the face of substantial and apparently increased ProDH activity within the bacteroids (Table I, Fig. 1 B). In bacteroids, proline accumulation was linear for 2 h. The total amounts of proline in bacteroids at times zero, 2 h, and 24 h were 90, 598, and 712 nmol/g fresh weight nodule, respectively. Because bacteroids occupy only about 20% of

0 .0

o Uq

puI"

-m- host cells | _~- bacteroids|

o

;

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5--.

4

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200 i

0*

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I 1

I I2 2

r

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I2 24

Time of incubation with added proline (h) Figure 2. Proline content of 35-d-old intact soybean root nodules as a function of time after supplying L-proline. Soybean plants were fed 20 mM L-proline solution (500 mL/20 cm diameter pot) at t = 0 and t = 12 h before harvesting. The amount of proline in bacteroids and host cells was determined by HPLC as described in "Materials and Methods." Error terms are ± 1 SE of three HPLC injections of preparations pooled from six or seven plants.

1 025


possibly because the partial pressure of 02 under aerated conditions may have been substantially higher than it was in

80(

°

potted greenhouse plants. If this is the case and [02] limits ProDH activity, then aeration might lead to a smaller proline

I*Eshost cells I O

D

a

0

bacteroidsl

60C

pool size in bacteroids.

A longer-term (24 h) incubation in aerated proline solution undertaken to assess the metabolic fate of ['4C]proline that reached the interior of nodules, as well as to estimate the was

40(

(presumably) steady-state value of proline resulting from the

CL

20

combined

impact of uptake and disappearance (export plus

metabolism). Ideally, we would have liked to have captured all metabolic products, but it is not possible to estimate the quantity of ["4C]proline converted in bacteroids 0.0

0.5

2.0

1.5

1.0

because respired CO2

can

originate from

any

to

14C02

soybean tissue.

However, in comparable experiments with isolated bacteroids,

Time h -

about 20% of the

[14C]proline taken up was recovered in CO2

(data not shown). Table III shows that, after 24 h of incubation in ["'C]proline, a significant portion of radiolabel was found

Figure 3. 14C accumulation in 35-d-old intact soybean root nodules as a function of time after supplying [J-14C]L-proline. Plants were gently uproote Idfrom perlite pots and placed in 50-mL test tubes with 20 mL 20 mM [U_14C]L-proline (5.5 uCi/mmol), and incubated for the indicated timets. Oxygen was supplied by bubbling air through the solution. Noduiles from three plants were pooled. Radioactivity was determined in ttriplicate. Error terms are ± 1 SE.

in the 70% ethanol-insoluble fraction, with the about twice

cytoplasm (17 is evidenced

high in bacteroids (29

as ±

not

2%). That proline only by the

was

±

2%)

percentage in host

as

actively metabolized

appearance

of radiolabel in

insoluble fractions, but is also consistent with the finding that

only a portion of the radioactivity in the soluble fractions was in proline (Table IV). Significant percentages of the radioactivity were present in organic and acidic amino acids and in P5C, with a higher proportion of the former in the host

Uptake of [UJ-'4CJProline into Soybean Nodule Fractions

cytoplasm and a higher proportion of the latter in bacteroids. A straightf orward interpretation of the data in Figure 2 is of radioactivity found in P5C in The significant that proline v was taken up by roots and moved into host cells higher in bacteroids, is host cells, with the and then intl wbacteroids. However, these data do not rigorrproline entering significant ously exclude e the possibility that the proline found in bbacterto P5C, and then exported dehydrogenated being bacteroids, ' oids was syn thesized there. To examine this possibility, we *[4C]proline mm, to host cytosol. Working in concert with high P5C reductase studied the fa activity of host cytosol (20), this would constitute a proline/ medium mL of aerated 5.5 ACi/mm(al))iincludedd inin 20 P5C shuttle of the type demonstrated in a cell-free rat liver ed medi.n 20 cus The time system containing mitochondria and extramitochondrial in Figure atiesonserven "pCr courthesameof shares some of the same propertes observed nmaterial (14). percentage

bactherpoidssibility

thesprolinedatdoud inot

even

consistent with a

percentage fraction of the

(20

c

aespite tne

very

aitterent expenmentl conaLiiLons; nameiy,

uprooted intact plants incubated with their roots in aerated proline solution in a laboratory hood under artificial illumination (Fig. 3) versus potted plants in the greenhouse irrigated with proline solution (Fig. 2). Uptake of proline into host cytoplasm was linear for 1 h, whereas bacteroids accumulated proline linearly for 2 h. However, unlike the results with the undisturbed greenhouse plants, the amount of proline in bacteroids never exceeded that in the cytoplasmic fraction,

Uptake of [5-3H]Proline into Soybean Nodule Fractions Although we believe the above data strongly support import of '4C as proline into the bacteroid from the host cell, the possibility, no matter how remote, exists that ["'C]proline found in bacteroids was synthesized there from more oxidized precursors generated in the cytoplasm and imported into bacteroids. To examine this possibility, we studied the fate of

Table Ill. Distribution of Radioactivity within Host Cells and Bacteroids Isolated from Nodules of 35-dold Soybean Plants Incubated in [U-'4C]Proline (5.5 MCiImmol) Plants were gently uprooted from perlite pots and placed in 50-mL test tubes with 20 mL of 20 mm proline and incubated for 24 h prior to harvest. Oxygen was supplied by bubbling air through the solution. Nodules from three plants were pooled. Radioactivity was determined in triplicate. Error terms

are+1 SE. Radioactivity 70% ethanol-insoluble 70% ethanol-soluble

Percentage Radioactivity in Ethanol-Insoluble Fraction

cpm/g fresh wt nodule

Cytosol Bacteroid

17871 ± 1797 9375 ± 875

3503 ± 208 3735 ± 188

16.6 ± 1.7 28.7 ± 1.5

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Table IV. Proline Metabolism in Host Cells and Bacteroids Isolated from Nodules of 35-d-old Soybean Plants Plants were incubated with 20 mL of 20 mm [U-14C]proline (5.5 ,ACi/mmol) 24 h prior to harvest. Error terms are ± 1 SE of triplicates samples. Calculated PretginPercentage i Fraction Radioactivity Organic and Acidic innPretgas Perolicnte P5C as Amount of Proline Amino AcidsPrsn cpm/g fresh wt nmol/g fresh wt Hostcells 17871 ± 1797 33.5 ± 1.2 55.4 ± 2.3 11.2 ± 1.2 818 ± 89 Bacteroids 9375 ± 875 16.2 ± 0.8 62.2 ± 0.4 21.6 ± 0.8 482 ± 45

exogenously supplied [5-3H]L-proline, because all of the 3H would be lost to the medium in the cytosol if it were oxidized to the level of glutamate or beyond before being imported into bacteroids and, according to the hypothesis being tested, reduced in that compartment to proline. Plants were incubated with [5-3H]L-proline (20 mm, 8.9 ,uCi/mmol) in 15 mL of aerated medium for 2 h. In contrast with the longer-term experiment with ['4C]proline, very little radioactive P5C was found in host cells in this short-term experiment, but a significant amount was found in bacteroids (Table V). We found a significant amount of [3H]proline in both host cytoplasm and bacteroids of soybean nodule preparations (Table V). Because one of the H atoms at C-5 of proline is lost to the medium when proline is dehydrogenated to P5C and the other is lost to the medium when P5C is dehydrogenated to glutamate, [3H]proline found in the bacteroid cannot have been synthesized from glutamate or a more oxidized precursor, such as succinate. The only escape from the conclusion that the [3H]proline found in the bacteroids is the result of [3H]proline crossing the peribacteroid membrane is to suggest that [3H]proline was dehydrogenated in the cytosol to P5C, with the P5C (which would retain part of the 3H originally in the proline) entering the bacteroid, where it could be reduced to proline. Disregarding the possible impact of the kinetic isotope effect on the interpretation of the data, half of the proline following this pathway would presumably contain 3H because the 3H in the added [3H]proline is almost equally distributed in the two sterically nonequivalent positions at C5 of proline. Thus, only half of the 3H would be lost in a cytosolic dehydrogenation of [5C-3H]proline, leaving open the possibility that [3H]P5C might move from the cytosol into

the bacteroid and there be reduced to [3H]proline. This seems unlikely for three reasons. First, proline supplied to isolated bacteroids is dehydrogenated (Table I, Fig. 1B). Second, stimulation of ARA by proline feeding (Table I) is inconsistent with reduction of P5C (an energy-requiring process) within the bacteroid. That is, for proline metabolism to enhance ARA, proline metabolism must be poised in an oxidative mode in the bacteroid to produce ATP and reductant needed for N2 fixation. Finally, the amounts of proline present in nodule fractions after feeding plants with [5-3H]proline, calculated from the radioactivity of proline isolated from these fractions, were very similar to those calculated from results of an experiment in which [U-'4C]proline was fed under identical conditions. The amounts calculated from 3H data were 291 ± 11 and 677 ± 83 nmol/g fresh weight for bacteroids and host cells, respectively (Table V). The corresponding amounts calculated from 14C data were 351 ± 10 and 653 ± 67 (Fig. 3). Had proline been synthesized from P5C within the bacteroids, the amount of proline calculated from 3H data should have underestimated the true amount by a factor of 2, since half of the tritium present in [5-3H]proline is lost when proline is dehydrogenated.

GENERAL DISCUSSION The accumulation of proline in bacteroids (Fig. 2) requires the rate of import of proline to exceed its rate of utilization. The data do not allow us to make estimates of the utilization rate in vivo that are reliable enough to calculate meaningful rates of proline import, because we have no measure of in vivo proline utilization by bacteroids. The rates calculated

Table V. Uptake and Metabolism of Radioactivity within Host Cells and Bacteroids Isolated from Nodules of 35-d-old Soybean Plants Incubated in [5-3H]L-Proline (8.9 AiCi/mmol) Plants were gently uprooted from perlite pots and placed in 50-mL test tubes with 15 mL of 20 mM aqueous L-proline and incubated for 2 h prior to harvest. Oxygen was supplied by bubbling air through the solution. Nodules from three plants were pooled and radioactivity counted as one group. Error terms are ± 1 SE of three experiments. Percentage as Percentage as Calculated Amount Calculated Amount Fro Rt Fraction Radioactivity Proline P5C of Proline Present of P5C Presenta cpm/g fresh wt nmol/g fresh wt Host cells 12103 ± 1636 99.0 ± 0.4 1.0 ± 0.4 677 ± 83 12.2 ± 5.6 Bacteroids 5869 ± 238 78.9 ± 1.1 11.8 ± 0.7 291 ± 11 77.7 ± 5.2 a One of the two H atoms at the carbon 5 position of proline is lost in the dehydrogenation of proline to P5C. This was taken into account in calculating the amount of P5C present.


from ProDH assays (based on the rate of loss of 3H from [SC3H]proline) are not a reliable indicator of absolute in vivo rates for at least two reasons: (a) depending on the details of the mechanism for dehydrogenation of proline, the uncorrected rates based on the "3H-loss" method published here and elsewhere might easily require an upward adjustment of a factor of 10 due to the 3H kinetic isotope effect; and (b) a more rapid turnover of the electron transport chain in vivo might reduce the impact of any rate limitation caused by the lesser availability of electron acceptors in the assay with isolated bacteroids. The important result from our experiments is that proline treatment had a physiological impact on processes that take place within bacteroids; namely, the accumulation of proline in bacteroids, the activity of ProDH, and the rate of acetylene reduction. This is the case whether or not the rate of proline entry in vivo is reasonably estimated by its rate of entry into isolated peribacteroid units (about one-third that of malate or one-tenth that of succinate, depending on the choice of substrate concentration at which the comparison is made [9]). Proline accumulation in soybean nodules and in bacteroids isolated from those nodules is not limited to circumstances in which high concentrations ofproline are supplied to the plant, as in the experiments whose results are reported here. Host cells and bacteroids from nodules of greenhouse-grown, drought-stressed 55-d-old plants accumulated 3.8 and 4.0 times the amount of proline, respectively, found in wellwatered controls (21). Proline accumulated to this extent in bacteroids despite a 1.6-fold increase in the assayed ProDH activity (21). The quantity of proline found in nodules of drought-stressed plants was 692 ± 71 (in the host cytoplasmic fraction) and 392 ± 18 nmol/g fresh weight (in the bacteroids). These values are of the same order as those reported here for nodule fractions of plants whose roots were bathed in 20 mM proline (545 ± 42 in host cells and 712 ± 28 nmol in bacteroids/g fresh weight of nodules collected from plants fed 20 mM proline at 24 h [Fig. 2]), indicating that the proline treatment was not so extreme as to render the results physiologically meaningless. CONCLUSIONS 1. Supplying proline to intact soybean plants stimulates ARA to the same extent as supplying succinate (Table I). 2. Application of proline causes an increase in ProDH activity (Table I and Fig. 1B), and the increase in ProDH activity is highly correlated with the increase in ARA (Fig. 1). 3. Despite the increase in ARA when proline is supplied to the root system of intact plants, proline application does not rescue stem-girdled plants from loss of ARA, although supplying succinate does (Table II). This suggests a nonphloem route of succinate, but not proline, from roots to nodules. 4. Applied proline accumulated in host cell cytoplasm and bacteroids to high levels (about three- and eightfold higher than controls) (Fig. 2). After proline feeding, proline concentration in bacteroids increased to at least 3.5 mm (calculated from data in Fig. 2). 5. Feeding [U-'4C]proline to intact plants resulted in accumulation of [14C]proline and products of proline metabolism in both host cells and bacteroids (Table IV). The significant

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percentage of radioactivity found in P5C in host cells, with the even higher percentage in bacteroids, is consistent with a significant fraction of the proline entering bacteroids, being dehydrogenated to P5C, and then exported to host cytosol, and with the operation of a proline/P5C shuttle. 6. Feeding [5-3H]proline also resulted in accumulation of labeled proline in both host cells and bacteroids (Table V). This could only result from [3H]proline or [3H]P5C entering bacteroids, because oxidation beyond P5C in either compartment would result in the loss to water of all 3H. For the latter route to contribute to the accumulated [3H]proline, [3H]P5C would have to be reduced within bacteroids. In contrast with the longer-term experiment (24 h) with [14C] proline, very little radioactive P5C was found in host cells in this 2-h experiment, but a significant amount was found in bacteroids. 7. The data reported here suggest a significant role for proline in bacteroid metabolism. Our working hypothesis is that this role is magnified during drought stress when proline accumulates in nodules in both host cells and bacteroids (21). ACKNOWLEDGMENTS We thank Karel Schubert, Oscar Chilson, and Fraser Bergersen for many helpful discussions about the work and the manuscript. We are especially grateful to Janet Sprent for the many revisions to the manuscript she suggested and to an anonymous reviewer whose hard questions made this a better paper than it otherwise would have been. LITERATURE CITED 1. Appleby CA (1984) Leghemoglobin and rhizobium respiration. Annu Rev Plant Physiol 35: 443-478 2. Arwas R, McKay IA, Rowney FRP, Dilworth MJ, Glenn AR (1985) Properties of organic acid utilization mutants of Rhizobium leguminosarum strain 300. J Gen Microbiol 131:

2059-2066 3. Bergersen FJ (1982) Root Nodules of Legumes: Structure and Functions. John Wiley & Sons, New York 4. Bergersen FJ, Turner GL (1988) Glutamate as a carbon source for N2-fixing bacteroids prepared from soybean root notdules. J Gen Microbiol 134: 2441-2448 5. Bergersen FJ, Turner GL (1990) Bacteroids from soybean root nodules: respiration and N2-fixation in flow-chamber reactions with oxyleghaemoglobin. Proc R Soc Lond B Biol Sci 238: 295-320 6. Bergersen FJ, Turner GL (1990) Bacteroids from soybean root nodules: accumulation of poly-f3-hydroxybutyrate during supply of malate and succinate in relation to N2-fixation in flowchamber reactions. Proc R Soc Lond B Biol Sci 240: 39-59 7. Birkenhead K, Sundaram SM, O'Gara F (1988) Dicarboxylic acid transport in Bradyrhizobium japonicum: use of Rhizobium meliloti dct gene(s) to enhance nitrogen fixation. J Bacteriol 170: 184-189 8. Chang J-Y, Knecht R, Braun DG (1983) Amino acid analysis in the picomole range by precolumn derivatization and highperformance liquid chromatography. Methods Enzymol 91: 41-49 9. Day DA, Ou-Yang LJ, Udvardi MK (1990) Nutrient exchange across the peribacteroid membrane of isolated symbiosomes. In P Gresshoff, E Roth, G Stacey, W Newton, eds, Nitrogen Fixation: Achievements and Objective. Chapman and Hall, New York 10. Day DA, Price GD, Udvardi MK (1989) Membrane interface of the Bradyrhizobium japonicum-Glycine max symbiosis: peribacteroid units from soybean nodules. Aust J Plant Physiol 16: 69-84 11. Farnden KJF, Robertson JG (1980) Methods for studying enzymes involved in metabolism related to nitrogenase. In FJ

1028

12.

13. 14. 15.

16. 17.

18. 19.

20.

21.

22. 23. 24.

ZHU ET AL.

Bergersen, ed, Methods for Evaluating Biological Nitrogen Fixation. John Wiley & Sons, New York, pp 265-314 Finan TM, Wood JM, Jordan DC (1981) Succinate transport in Rhizobium leguminosarum. J Bacteriol 148: 193-202 Fitzmaurice A, O'Gara F (1990) Involvement of glutamate as a carbon source in nitrogen-fixing Rhizobium meliloti. Biochem Soc Trans 18: 358-359 Hagedorn CH (1986) Demonstration of a NADPH-linked A'pyrroline-5-carboxylate-proline shuttle in a cell-free rat liver system. Biochim Biophys Acta 884: 11-17 Hagedorn CH, Phang JM (1986) Catalytic transfer of hydride ions from NADPH to oxygen by interconversion of proline and A'-pyrroline-5-carboxylate. Arch Biochem Biophys 248: 166-174 Hardy RWF, Holsten RD, Jackson EK, Burns RC (1968) The acetylene-ethylene assay for N2 fixation. Laboratory and field evaluation. Plant Physiol 43: 1185-1207 Heckmann M-O, Drevon J-J, Saglio P, Salsac L (1989) Effect of oxygen and malate on NO3- inhibition of nitrogenase in soybean nodules. Plant Physiol 90: 224-229 Herrada G, Puppo A, Rigaud J (1989) Uptake of metabolites by bacteroid-containing vesicles and by free bacteroids from French bean nodules. J Gen Microbiol 135: 3165-3171 Kahn ML, Kraus J, Somerville JE (1985) A model of nutrient exchange in the Rhizobium-legume symbiosis. In H Evans, P Bottomley, W Newton, eds, Nitrogen Fixation Research Progress. Martinus Nijhoff, Dordrecht, pp 193-199 Kohl DH, Schubert KR, Carter MB, Hagedorn CH, Shearer G (1988) Proline metabolism in N2-fixing root nodules: energy transfer and regulation of purine synthesis. Proc Natl Acad Sci USA 85: 2036-2040 Kohl DH, Kennelly EJ, Zhu Y-X, Schubert KR, Shearer G (1991) Proline accumulation, nitrogenase (C2H2 reducing) activity, and activities of enzymes related to proline metabolism in drought stressed soybean nodules. J Exp Bot 42: 831-837 Magasanik B (1982) Genetic control of nitrogen assimilation in bacteria. Annu Rev Genet 16: 135-168 Phang JM (1985) The regulatory functions of proline and pyrroline-5-carboxylic acid. Curr Top Cell Regul 25: 91-132 Phang JM, Downing SJ, Valle DL, Kowaloff EM (1975) A radioisotopic assay for proline oxidase activity. J Lab Clin Med 85: 312-317


25. Raven JA, Sprent JI, Mclnroy SG, Hay GT (1989) Water balance of N2-fixing root nodules: can phloem and xylem transport explain it? Plant Cell Environ 12: 683-688 26. Ronson CW, Lyttleton P, Robertson JG (1981) C4-dicarboxylate transport mutants of Rhizobium trifolii form ineffective nodules on Trifolium repens. Proc Natl Acad Sci USA 78: 42844288 27. Salminen SO, Streeter JG (1987) Involvement of glutamate in the respiratory metabolism of Bradyrhizobiumjaponicum bacteroids. J Bacteriol 169: 495-499 28. Schubert KR (1982) The Energetics of Biological Nitrogen Fixation. Workshop Summaries-I. American Society of Plant Physiologists, Rockville, MD, pp 1-30 29. Stewart CR (1981) Proline accumulation: biochemical aspects. In L Paleg, D Aspinall, eds, Physiology and Biochemistry of Drought Resistance in Plants. Academic Press, Sydney, pp 243-259 30. Udvardi MK, Price GD, Gresshoff PM, Day DA (1988) A dicarboxylate transporter on the peribacteroid membrane of soybean nodules. FEBS Lett 231: 36-40 31. Udvardi MK, Salom SA, Day DA (1988) Transport of L-glutamate across the bacteroid membrane but not the peribacteroid membrane from soybean root nodules. Mol Plant-Microbe Interact 1: 250-254 32. Walsh KB, Canny MJ, Layzell DB (1989) Vascular transport and soybean nodule function. II. A role for phloem supply in product export. Plant Cell Environ 12: 713-723 33. Walsh KB, McCully ME, Canny MJ (1989) Vascular transport and soybean nodule function: nodule xylem is a blind alley, not a throughway. Plant Cell Environ 12: 395-405 34. Witty JF, Minchin FR, Skot L, Sheehy JE (1986) Nitrogen fixation and oxygen in legume root nodules. In F Minchin, ed, Oxford Surveys of Plant Molecular and Cell Biology. Oxford University Press, Oxford, pp 275-314 35. Wong PP, Evans HJ (1971) Poly-,B-hydroxybutyrate utilization by soybean (Glycine max Merr.) nodules and assessment of its role in maintenance of nitrogenase activity. Plant Physiol 47: 750-755 36. Zhu Y-X, Schubert KR, Kohl DH (1991) Physiological responses of soybean plants grown in a nitrogen-free or energy limited environment. Plant Physiol 96: 305-309