PI VSIOL, PLANT. 61: 637-642. Copenhagen 1984

Growth and root nodule nitrogenase activity oi Pisum sativum asinfluenced by transpiration

Ries de Visser and Hendrik Poorter

de Visser, R. and Poorter, H. 1984. Growth and root nodule nitrogenase activity ofPisum sativum as influenced by transpiration. - Physiol. Plant. 61: 637-642.

The influence of shoot transpiration on the rates of growth and nitrogen fixation wasinvestigated in Pisum sativum L. cv. Rondo. In short term experiments, rates oftranspiration and acetylene reduction of intact plants were measured simultaneously,using air-tight perspex vessels enclosing the basal part of the nodulated root. In longterm experiments, accumulation of dry matter and reduced nitrogen in the plant weredetermined as well. Transpiration rate changed diurnally and was varied by manipu-lating the vapour saturation deficit or the flow rate of the air in the growth cabinet.The rate of acetylene reduction declined after subjecting intact plants to high trans-piration rates. This decline was accompanied by a desiccation of the root nodules.Dry matter and reduced nitrogen accumulation were not affected by transpirationrate. At low transpiration rate reduced nitrogen content of the root nodules washigher than at high transpiration rate. However, in these nodules the rate of acety-lene reduction was not significantly affected. It is concluded that the nitrogenaseactivity of pea root nodules is insensitive to changes in the flow rate and the organic Nconcentration of the xylem sap within a wide range of transpiration conditions underthe applied growth conditions.

Additional key words - Acetylene reduction, nitrogen accumulation, nitrogen fixa-tion, pea.

R. de Visser (reprint requests), Dept of Plant Science, Univ. of Alberta, Edmonton,Alberta, Canada T6G 2P5; H. Poorter, Dept of Plant Physiology, Univ. of Gro-ningen, P.O. Box 14, 9750 AA Haren, The Netherlatids.

Introduction

The symbiotic associations of leguminous plants andRhizobium bacteria are able to reduce atmospheric ni-trogen to ammonium in their root nodules. The reduc-tion is catalysed by nitrogenase in the bacteroids, whichexcrete the ammonium into the plant cell cytoplasmwhere it is incorporated in amides and amino acids.These are loaded into the xylem and transported to theshoot via the transpiration stream (Minchin and Pate1974, Pate 1976, Shanmugam et al. 1978). The activityof nitrogenase has been suggested to be regulated by theenergy supply of the root nodules (M. van Mil 1981.Thesis. Univ. of Wageningen, The Netherlands; for areview see Minchin et al. 1981) and by end products ofnitrogen fixation (e.g. ammonium; O'Salminen 1981

and references therein). In contrast to the regulation byenergy supply, end product inhibition has not been in-tensively studied. O'Salminen (1981) reported inhibi-tion of acetylene reduction by ammonium in cell-freeextracts of root nodules of Pisum sativum. In a shortterm experiment with Lupinus albus, Lambers et al.(1980) found that the rate of acetylene reduction in-creased at high rates of shoot transpiration which arelikely to enhance the removal of fixation products.

Minchin and Pate (1974) did not find a correlationbetween transpiration and acetylene reduction rate ofpea root nodules although the concentration of solublenitrogen in the nodules increased at low transpirationrates. However, long term effects of transpiration onfixation and accumulation of nitrogen have not beeninvestigated in plants not suffering water stress.

Received 31 October, 1983; revised 26 March, 1984

Phvsiu Plant. 61 . 19S4 637

In the present paper, we examine short as well as longterm effects of transpiration on nitrogenase activity ofroot nodules in Pisum sativum exploring a wide range oftranspiration rates as found during a diurnal cycle(Minchin and Pate 1974) or created by varying flow rateand humidity of the air (Poljakoff-Mayber and Gale1972).

Abbreviation - VSD, water vapour saturation deficit.

Materials and methods

Plant material

Seeds of Pisum sativum L. cv. Rondo were surfacesterilized, germinated in the presence of Rhizobiumleguminosarum strain S313 (Lab. of Microbiology, Wa-geningen; a strain producing large, branched nodules)and grown on continuously aerated nutrient solutions asdescribed by de Visser and Lambers (1983). Germina-tion and growth occurred in a growth chamber at aconstant temperature of 20°C (18°C in experiment I, seebelow). The water vapour saturation deficit (VSD) andthe flow rate of the air were controlled at differentlevels, depending on the experiment (see below). Lightintensity was 250 jxmol m"- s"' (photosynthetically activeradiation), supplied by Philips HPL 450 W and incan-descent lamps (100 W) in a ratio of three to twoprovided with a water cuvette, during 16 h per day.During the second week after sowing all secondaryroots along the first 7 cm of the basal part of the mainroot were removed successively to facilitate handlingand prevent rapid oxygen depletion during the acety-lene reduction measurements (Dart and Day 1971).

Harvest

After the measurements of transpiration and acetylenereduction, plants were separated into shoots, roots andnodules. Fresh weight and leaf area were determined.Plant material was dried overnight at 90°C and dryweight was determined.

Simultaneous measurement of acetylene reduction andtranspiration

The basal parts (7 cm) of the main roots of two nodul-ated plants were enelosed in a perspex cuvette with avolume of 220 ml and provided with a rubber septum(Suba-Seal). The cuvette was placed on a PVC vessel of0.5 1 containing a nutrient solution of the same composi-tion as during growth. The solution was continuouslyaerated with water saturated air. After a 1 h acclimationperiod, measurements of transpiration (gravimetrieally)and acetylene reduction were started. Acetylene wasinjected into the cuvette giving a partial pressure of 7kPa.

20 40time in min

Fig. 1. Time course of ethylene production by roots of Pisum.sativum L. cv. Rondo nodulated with R. leguminosarum strainS313. The amount produced after 1 h incubation is set at 100%.Mean ± SE, n = 6. The dotted line represents an example ofethylene production by legumes, as observed by Minchin et al.(1983); recalculated data).

During the subsequent 1.5 h, 4 samples of 500 1̂ weretaken with a gas tight syringe and injected into glassvials of 2,5 ml, which were stored for 2 h. Following theharvest of the plants, ethylene and acetylene were de-termined by gas chromatography using a flame ioniza-tion detector mounted on a Packard model 421 BeckerGas Chromatograph. A glass column (length 1.2 m, 0 2mm) filled with 100-120 mesh Porapaek R was used.The column temperature was 50°C, the detector tem-perature was 120°G. The output signals were processedby a Varian CDS 111 integrator. Root nodule ethyleneproduction was linear with time (Fig. 1), in accordancewith data of Minchin et al. (1983) on peas grown at a16 h photoperiod. Measurements never started before5 h after the dark period.

Leaf area

The total area of the leaf blades and stipules of the twoplants used was determined photometrically with aLi-Cor model 3100 area meter (LI-COR, Inc., Lincoln,NE).

Reduced nitrogen content

Total content of reduced nitrogen was determined onpulverized dry material with the Kjeldahl method(Bailey 1962), using K^SOj and CuSOj in a ratio of 3:1(w/w) as a catalyst.

Statistical inference

Data were evaluated by one- or two-factorvariance, or by Student's /-test.

Nis of

638 Physiol. Plant. . I')S4

E periments

S oot transpiration rate was recorded diurnally and wasmanipulated in two other experiments by varying thefl' 'W rate or the vapour pressure deficit of the air (Polja-k(iff-Mayber and Gale 1972).

Exp. 1. The diurnal course of transpiration andm etylene reductionPlants were used 24 days after sowing. VSD was 9.6 gm \ Six sets of two plants were taken at 5, 9, 14, 18, 22and 38 h after the start of the photoperiod.

Exp. II. Transpiration and N^-fixation at two air flowratesWithout any change in other growth conditions, trans-piration was gradually enhanced by increasing the airflow rate from 0.2 to 2.0 m s"'. Three weeks after sowingone half of the plants was switched to the higher air flowrate, generated by two ventilators. VSD of the air was7.0 g m~\ Transpiration and acetylene reduction weremeasured in quadruple, simultaneously on plants ofhoth treatments, at 1, 5, 25, 49 and 97 h after startingthe treatment.

Exp. III. Transpiration and N2-fixatiort at two airhutniditiesSince air flow rate may affect photosynthesis, or mor-phology (Whitehead 1962), transpiration was loweredby enhancing the air humidity. VSD was 9.6 g m"̂ .Twenty days after sowing one half of the plants wastransferred to a climate chamber with the same condi-tions, except for the VSD, which was kept constant at1.7 g m' \ Measurements were made in quadruple at 1 hand 1, 2, 5, 8 and 12 days after the switch, simul-taneously on both groups of plants. A reciprocal shortterm switch was conducted 6 days after the start of thetreatment.

Results

Diurnal course of transpiration and acetylene reduction

Figure 2 shows a steadily decreasing transpiration rateduring the light period {P < 0.01), probably caused bythe high VSD (9.6 g m •̂ ). Differences were also foundhetween the light and the dark period {P < 0.001). Rootnodule acetylene reduction rate showed fluctuationswhich were not significant (Fig. 2). The same applies tothe differenee in activity between the light and the darkperiod. The latter result may be attributed to the con-stant temperature during growth (Ryle et al. 1979).

Transpiration and Ni-fixation at different air flow rates

Figure 3 shows the time course of the transpiration rateof I'lants grown at two different air flow rates (0.2 and2.0 m s"'). Air flow rate effects on transpiration rate

38

Fig. 2. Diurnal course of shoot transpiration rate (O) and rootnodule acetylene reduction rate ( • ) of pea cv. Rondo {Pisumsativum L.) nodulated with Rhizobium leguminosarum strainS313 (lacking Hup). Mean ± SE, n = 6.

were significant, on a plant basis (results not shown; P< 0.001) as well as on a leaf area basis {P < 0.001). Oneday after the switch of the high air flow plants back to a

time in days

Fig. 3. Time course of shoot transpiration rate of nitrogenfixing plants of Pisum .sativum L. cv. Rondo grown at high (2.0m s-i, O) and low (0.2 m s '̂, • ) air flow rate. Time in days afterstarting the high air flow treatment. The arrow indicates theend of the high air flow treatment on day 4. Mean ± SE, n = 4.

time in days

Fig. 4. Root nodule acetylene reduction rate of pea plants{Pisum .sativum L. cv. Rondo) grown at high (2.0 m s"', O) andlow (0.2 m s"', • ) air flow rate. The arrow indicates the end ofthe high air flow treatment on day 4. Time in days after startingthe high air flow treatment. Mean ± SE, n = 4.

liys; !. Plant. 61, 1984 639

Tab. 1. Relative growth rate (RGR) and reduced nitrogencontent and accumulation in pea plants fixing dinitrogen athigh or low air flow rate. Mean ± SE, n= 4.

Air flow ratem s"'

0,22.0

RGRday-'

0.0640.064

Reduced

Contentmg N g-'

39.1 ±1.8438.2±0.67

nitrogen

Accumulationmg N plant-'

13.4±2.2113.7±0.87

low air flow rate, transpiration rate was lower than inthe eontrol (F < 0.05). Total leaf area per plant was notinfluenced by air flow rate (results not shown). Theacetylene reduction rate of the nodules (on a dry weightbasis) was not changed by air flow rate (Fig. 4). Therelative growth rate over the 5-day experimental periodwas the same at both air flow rates (Tab. 1). After fourdays still no difference was found in the content oraccumulation of nitrogen (Tab. 1).

DOO

^ 5 0 0Q.O)

J.400_g)

^300-a

roD.

200_

: J1 sJr

^ 1

' I 1 1 1

4 8time in days

12

Fig. 6. Dry matter accumulation of nitrogen fixing pea plants{Pisum sativum L. cv. Rondo) grown at low (1.7 g m-3, • ) andhigh (9.6 g m-3, O) VSD. Note the log-scale of the y-axis. Timein days after switching one half of the plants to low VSD.Broken lines indicate the short term (1 h) switches. Mean ± SE,n = 4.

IVanspiration and Nj-fixation at two air humidities

Figure 5 shows that transpiration rate declined (P <0.001) upon decreasing the VSD to 1.7 g m'l However,leaf area per plant did not change (results not shown) incontrast with data of Poljakoff-Mayber and Gale(1972). The same difference in transpiration rate wasfound in short term experiments (Fig. 5; reciprocalswitch). Dry matter accumulation (Fig. 6) was not af-fected by VSD within 12 days, neither was the shoot toroot ratio (results not shown). This agrees well withdata of Lambers and Posthumus (1980) on maize. Theacetylene reduction rate of the nodules was increased byea 35% during five days after the switch to low VSD(Fig. 7A). Moreover, short term switches resulted inhigher activities when switching to low VSD (Fig. 7A; P< 0.05; reciprocal switch).

During the acetylene reduction assay, dry matter con-tent of fresh weight of the root nodules in the perspex

CL

4 -

_

'• . 1

1111

fI

time in days12

Fig. 5. Rate of shoot transpiration of nitrogen fixing pea plants{Pisum sativum L. cv. Rondo) grown at low (1.7 g m '̂, • ) andhigh (9.6 g m-', O) VSD. Time in days after switching one halfof the plants to low VSD. Broken lines indicate the short term(1 h) effect of a reciprocal switch on transpiration rate. Mean ±SE, n = 4.

time in days12

Fig. 7. Root nodule acetylene reduction rate (A) and rootnodule dry matter content (B) of pea plants {Pisurn sativum L.cv. Rondo) grown at low (1.7 g m--\ • ) and high (9.6 g m •', 0)VSD. Time in days after switching one half of the plants to lowVSD. Broken lines indicate the short term (1 h) effect of areciprocal switch on acetylene reduction rate and on noduledry matter content. Mean ± SE, n = 4.

640 P h y s i o l . P l a n l . ••!. 1984

Ti 1. 2. Leaf area (cm- plant-') and reduced nitrogen accumu-la ed (mg N plant-') of nitrogen fixing pea plants grown duringl: days at low (1.7 g m-') or high (9.6 g m-') VSD. Treatmentst rted 21 days after sowing (day 0). Mean + SE, n = 4.

T; ne

d;)'s

058

12

Leaf

low VSD

41.5±1.570.3±7.586.5±8.7

119.0±6.5

area

high VSD

44.0± 4.876.8+ 4.484.4± 6,1

116.3±15.3

Reduced N

low VSD

8.5±0.313.0±1.815.6+2.022.4+1.7

accumulated

high VSD

9.5±0.613.2±0.715.5+1.021.2+3.7

Tab. 3. Accumulation of reduced nitrogen in the root nodulesof plants of Pisum sativum grown at low (1.7 g m-') and high(9.6 g m-') VSD. Treatment started 21 days after sowing (day0). Mean ± SE, n= 4.

Time

days

058

12

Reduced(% of

low VSD

10.3±1.016.1±0.515.2±0.814.5±0.5

N intotal

root nodulesplant N)

high VSD

9.211.114.8±0.714.2±0.412.4±0.6

vessel was significantly affected by VSD, as demon-strated by the short term switch (1 h) to high VSD (P <0.01; Fig. 7B). Leaf area and reduced nitrogen ac-cumulation were not sensitive to changes in VSD (Tab,2). However, the amount of reduced nitrogen accumul-ated in the root nodules increased at low transpirationrates (Tab, 3; F < 0.05).

Discussion

Transpiration rate of the shoot was significantly influ-enced by the time of the day, air flow rate and waterVSD (Figs 2, 3, and 5), and ranged from 2.5 to 9.0 fxlH2O h"' cm"- leaf area, 5.5 being the commonly ob-served rate of transpiration at normal growth conditions(VSD 7 g m-\ 20°C, 0.2 m s"', 250 ^mol m"- s"'). Hightranspiration rates decreased the rate of acetylene re-duction during five days after the switch from low VSDto high VSD in experiment III (Fig. 7A).

The accumulation of dry matter and nitrogen was notinfluenced by transpiration rate in the same experiment(Fig. 6, Tab. 3). Cell growth (Hsiao 1973) and leafgrowth (Boyer 1970, Acevedo et al. 1971) are amongthe processes most sensitive to water stress. However,neither in the air flow experiment, nor in the air hu-midity experiment was leaf area per plant affected bytreatment (results not shown), indicating that the plantsdici not suffer water stress during growth. Nevertheless,root nodule acetylene reduction was stimulated duringODO to five days after subjecting the plants to a high air

humidity which decreases transpiration rate (Fig. 7A).The time course of root nodule dry matter content andthe reciprocal switch (Fig. 7B) demonstrate that rootnodules desiccated in the measuring vessel, unless theplants were kept under low transpiration conditions.The concomitant decrease in acetylene reduction rate(Fig. 7A) is in agreement with data of Sprent (1972).According to Pankhurst and Sprent (1975a), epidermiscells of the root nodules are loosely packed, guarantee-ing an optimal diffusion of gases. At increasing desicca-tion this loose package is maintained to a certain degreeof desiccation, after whieh the package becomes com-pletely closed, severely hampering the diffusion ofgases. Oxygen supply to the inner nodule seems to bethe most sensitive process. Pankhurst and Sprent(1975b) observed a recovery of the acetylene reductionrate after increasing the partial oxygen pressure aroundthe desiccated nodules (cf. Dart and Day 1971).

In the root nodules of Pisum sativum a dry mattercontent of 14% is suggested to be a critical value. Athigher dry matter contents acetylene reduction was de-pressed (Fig. 7). The root nodules did not desiccatewhen submerged in aerated nutrient solution. The mea-suring vessels always contained some water and wereclosed hermetically, assuring water saturation of the airinside. Apparently, root nodule functioning dependsdirectly on continuous water uptake by the nodule it-self, as long as transpiration by the shoot is substantial.This condition is incompatible with the requirements ofacetylene reduction measurements on intact plants, andis probably the cause of the rapid decline (within 10min) of acetylene reduction in open, continuous air flowsystems (Minchin et al. 1983; ef. Fig. 1). Moderatewater stress is known to be a major factor lowering therate of nitrogen fixation in the field in nodules near thesoil surface (Sprent 1971).

Root nodule dinitrogen fixation could not be stimu-lated by increasing the rate of transpiration (Fig. 3, Tab.1), as reported by Lambers et al. (1980). The data onthe accumulation of reduced nitrogen in the plants(Tabs 1 and 2) clearly demonstrate that root nodulenitrogen fixation was independent of the rate of shoottranspiration, although indeed more nitrogen was ac-cumulated in the nodules at low transpiration rates inexperiment III (Tab, 3), in accordance with results ofMinchin and Pate (1974) on Pisum sativum. It is there-fore concluded that under the present growth conditionsthe organic N concentration and flow rate of the xylemsap do not control the activity of nitrogenase.

Acknowledgements - We thank Dr Hans Lambers for valuablesuggestions, Dr Rinie Hofstra for stimulating discussions andcritical reading of the manuscript, Dr T. A. Lie for helpfulcommentary and for providing the Rhizobium bacteria and MrEvert Leeuwinga for drawing the figures. This research wassupported by the Foundation for Fundamental Biological Re-search (BION) which is subsidized by the Netherlands Organi-zation for the Advancement of Pure Research (ZWO).

'hysiol. Plant. 6t. 1984 641

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Edited by P. Nissen

642 Physiol. Plant. 6i 1984