PHYSIOLOGIA PLANTARUM 82: 5S1-5S8, Copenhagen 1991

Nitrate rediictase activity, nitrogenase activity and photosyntliesisof black alder exposed to chilling temperatures

Christoph S. Vogd and Jeffrey O. Dawsom

Vogel, C. S, and Dawson, J, O. 1991, Nitrate reductase activity, nitrogenase activityand photosynthesis of black alder exposed to chilling temperatures, - Physiol, Plant,82: 551-558,

Actinorhizal (/^ran/rifl-nodulated) black alder [Alnus glutinosa (L.) Gaertn,] seed-lings fertilized with 0,36 mM nitrate (low nitrate fertilizer treatment) or 7,14 mMnitrate (high nitrate fertilizer treatment) and acclimated in a growth chamber for 2weeks were exposed to 2.5 h of night-time chilling temperatures of —1 to 4°C. Coldtreatment decreased nitrogenase activity (acetylene reduction activity) 33% for lownitrate fertilized plants and 41% for high nitrate fertilized plants. Recovery ofnitrogenase activity occurred within 7 days after chilling treatment. In contrast, invivo nitrate reductase (NR) activities of leaves and fine roots increased immediatelyafter chilling then decreased as nitrogenase activities recovered. Fine roots of alderseedlings exhibited NR activities proportional to the amounts of nitrate in the rootingmedium. In contrast, the NR activities of leaves were independent of substrate andtissue nitrate levels and corresponded to nitrogenase activity in the root nodules. In aseparate experiment, net photosynthesis (PS) of similarly treated black alder seed-lings was measured before and after chilling treatments. Net PS declined in responseto ehiUing by 17% for plants receiving low nitrate fertilizer and 19% for plantsreceiving high nitrate fertilizer. After chilling, stomatal conductance (gj decreasedby 39% and intemal CO'2 concentration (c ) decreased by 5% in plants receiving thehigh nitrate fertilizer, whereas plants receiving the low nitrate fertilizer showed noebange in gs and a 13% increase in q. Results indicate that chilhng stimulates stomatalclosure only at the high nitrate level and that interference with bioehemtcal functionsis probably the major impact of chilling on PS,

Key words - Aetinorhizal, Alnus glutinosa, black alder, chilling temperatures, di-nitrogen fixation, Frankia, nitrate reductase, nitrogenase, photosynthesis.

C. S. Vogel and J. O. Dawson (corresponding author), Dept of Forestry, Univ. ofIllinois, 1301 West Gregory Dr., Urbana, IL 61801, USA.

ing temperatures began to occur and after the leaves ofIntroduction associated woody deciduous broad-leaved trees haveBlack alder \Alnus glutinosa (L.) Gaertn,] is a temper- been shed (Neave et al, 1989),ate deciduous tree that forms symbiotic dinitrogen fix- Leaf N resorption in black alder during the autumning root nodules in association with soil actinotnycetes can be affected by soil fertility and Frankia nodulation,of the genus Frankia. Black alder sheds its leaves much Cote et al. (1989) demonstrated that N-fertili2ed blacklater and resorbs less net leaf N during autumnal se- alder seedlings lacking Nj-fixing nodules showed no netnescence than do most other temperate deciduous trees resorption of leaf N during autumn, while alders de-(C6te and Dawson 1986, Gote et al, 1989, Dawson and pendent on N derived from symbiotic fixation resorbedFunk 1981, Dawson etal, 1980), This delay in autumnal 38% of their leaf N prior to leaf abscission. The declinesenescence apparently allows leaf physiological proc- in leaf N coincided with the temperature-dependentesses to continue longer, since photosynthesis (PS) in decrease in nitrogenase activity. Fertilized plants lack-black alder may cotitinue for up to a month after freez- irtg nodules continued to absorb and utilize available

Received 5 November, 1990; revised 26 March, 1991

Ptlysiol; Ptanl. 82,1991 5 5 1

soil N when soil temperatures were low enough to inhib-it nodular nitrogenase activity. The authors suggestedthat decreased N input to leaves due to decreased nitro-genase activity in the autumn was responsible for thenet decline in leaf N concentration.

Nitrate is the major form of plant-available N in manysoils, incltiding soils planted with actinorhizal black al-der trees (Paschke et al, 1989) and nitrate reductase is akey enzyme in the metabolism of nitrate by higherplants (Campbell 1988), Nitrogenase activity of blackaider seedlings and ramets is positively correlated witbphotosynthetic activity (Dawson and Gordon 1979,Gordon and Wheeler 1978), Thus, the impact of chillingon plant nitrogenase and possibly NR activities mayreflect its impact on photosynthetic activity. Cool tem-peratures can also increase plant root NR activity andrate of ion uptake (Clarkson and Deane-Drummond1983, Ingestad 1979), In this study we hypothesized thatchilling temperatures would inhibit nitrogenase activityof root nodules of black alder, but not the NR activitiesof roots and leaves, and that photosynthesis would beinhibited less than nitrogenase activity.

Abbreviations - Cj, Internal CO, concentration; g , stomatalconductance; NR, nitrate reductase; PAR, photosyntheticallyactive radiation (400-700 nm); PS, photosynthesis.

Tab, 1, Compositions of low nitrate and high nitrate fertilizers.

Component

Ca(NO,), • 4H,OKNO,KH,PO'4K,HPO4MgSOj • 7H,OCaCl, - 2H,OKCl

Final concentrations (mAf)

Low nitrate

0.18_

0.8640,1362,02,044,8

High nitrate

1.24.80.8640.1362.01,02

-

were maintained at pH 5,85 and contained full-strengthHoagland's micronutrients supplemented with 2 mg T'Fe as FeSO^-EDTA.

Three separate temperature treatments were initiatedby transferring growth-chamber-acclimated plants to arefrigerated room (Bangor Cooler Company, Hartford,MI, USA) 2 h after the lights were turned off for 2,5 h at4, 1 or — 1°C, Measurements in the soil indicated thatrhizosphere temperature equilibrated witb air temper-ature after 1,5 h. Immediately after cold temperaturetreatment, piants were returned to the growth chamber.Assays for NR, nitrogenase and photosynthetic activ-ities were performed 1 day prior to and 1, 4 and 7 daysafter temperature treatments.

Materials and methods

Plant culture and treatments

Half-sib seeds collected from naturalized black aldertrees growing in central Illinois, USA were sown insteam-pasteurized loam soil:peat:perlite (1:1:1, v/v/v) inthe greenhouse and inoculated with an homogenate ofFrankia isolate ArI3 (Berry and Torrey 1979) 4 weeksafter seed germination. Four weeks after inoculationseedlings were transplanted into 12,5-cm top diam, x12,5-cm deep plastic pots (750 ml vol,) filled with silicasand. Plants received 1/4-strength Hoagland's solution(Hoagland and Amon 1950) containing 5 mg 1~' N asammonium nitrate 3 times a week while in the green-house. Six- to 10-month-old experimental plants weremoved to a model E15 Conviron growth chamber (Con-trolled Environments Inc, Pembina, ND, USA) andacclimated for 2 weeks prior to imposition of temper-ature treatments. Plants were maintained in the growtbchamber at a 12-h photoperiod with 40 W Phillips coolwhite fluorescent bulbs and 25 W General Electric in-candescent bulbs together providing 480 |imol m^ s"'PAR at the top ofthe crown, and at 20°C day/15°C nighttemperatures and 50% relative humidity. Plants in thegrowtb chamber were randomly assigned to 2 equalgroups that received daily fertilizer treatments consist-ing of either 0,36 mM nitrate (low nitrate fertilizertreatment) or 7,14 mM nitrate (high nitrate fertilizertreatment), Macronotrient components of the 2 nitratefertilizers are given in Tab, 1. Both fertilizer solutions

Nitrogenase assay

Nitrogenase activity was estimated by the acetylene re-duction technique (Gibson 1987) on root systems ofintact plants in pots. The same 3 plants in each of thenitrate treatments were repeatedly assayed throughouta single temperature treatment period because consid-erable plant to plant variation existed in specific nitro-genase activity [nmol ethylene prodtieed (g nodule dryweight)"' h"'], Neave and Dawson (1989) showed thatthe specific nitrogenase activity of black alder seedlingsdid not change during a 1-week period of daily acety-lene reduction assays, so we were confident that acety-lene incubation would not have significant effects onnitrogenase activity of plants in the current experiment.

Plant root systems in their pots were sealed in 2,5-1plastic containers by placing over them a radially splitcover with a central hole that would accommodate thestem at approximately 2 cm above the root collar. Thecover was made gas-imperyious by applying plasticine-clay around the stem and along the radial seam, Avolume of air was removed by syringe from the in-cubation vessel through a serum stopper and replacedwith an equal volume of acetylene to make a 10%acetylene atmosphere. The assay was conducted in thegrowth chamber at 20°C for 1 h between hour 6 and 7 ofthe photoperiod, after which a 10-ml volume of gas wasremoved through the serum stopper and placed in gas-tight blood collection tubes (Vacutainer brand 6441,Becton Dickinson and Co,, Rutherford, NJ, USA) for

552 Pkysiot, Planl. 82, l

analysis of ethylene concentration by gas chromatog-raphy.

NR assay

In vivo NR activities of leaves and roots were per-formed using modifications of the method of Hagemanand Reed (1980), Leaf and root tissues of 3 plants fromeach of the 3 temperature treatments and 2 fertilizertreatments were harvested on the same days on whichnitrogenase activities were determined and between 8and 10 h into the photoperiod. Leaf samples weighing20 to 25 mg consisted of 10 5-mm diam, leaf discs, 2discs from each of the most recently fully expanded 5leaves. Root tissue selected for NR assays consisted ofpieces of fine roots (those occurring within 2 cm of theroot tips) that had total fresh weights of approximately20 mg. The fine roots had higher activity than olderroots or adventitous roots.

After all of tbe tissue samples had been collected andplaced in separate tubes, each containing 1 ml of icecold deionized H2O, 1-ml portions of double-strengthincubation medium were added to each tube containingharvested tissue. Leaf and root NR incubation mediawere optimized for pH, nitrate, phosphate and n-propa-not concentrations. The final concentrations of the com-ponents in the leaf incubation medium after 1:1 dilutionwith deionized HjO were 8 mM KH,PO4, 42 mMK,HPO4, 100 mM KNO3 and 2% «-propanol (v/v). Theroot incubation medium contained 9 mM KH2PO4, 42mM K2HPO4, 50 mM KNO3 and 2% «-propanoI (v/v).Both media were at pH 7,5. Forty-mesh wire screenswere placed in the tubes to hold the tissues below thesurface of the incubation medium and the tubes weretransferred to an Evapotech vortex-evaporator (Haake-Buchler Instoiments, Inc, Saddle Brook, NJ, USA),vacuum infiltrated twice at 91,2 kPa for 2 min andincubated at 30°C in the dark for 60 min at a vortexspeed of 60 rpm, NR activities of both roots and leaveswere linear during a time internal of 50 to 75 min afterinitiation of vacuum infiltration under the conditions ofour NR assay. Results from preliminary experimentsshowed that there was little NR activity associated withroot or leaf tissue if nitrate was not included in theincubation media, and nitrate was used ih the incuba-tion media at concentrations that resulted in maximal invivo NR activities. The samples were placed on iceimmediately after incubation and 250 1 from each ofthe samples were assayed for nitrite concentration bythe method of Keeney and Nelson (1982), All NR activ-ities were expressed on a tissue fresh weight basis,

PS assay

In a separate experiment, PS, g, and q were determinedfor black alder seedlings exposed to the same growthconditions, fertilizers and cold temperature treatmentsas described above. Assays were conducted using an

LCA-2 infra-red gas analyzer (The Analytical Devel-opment Company, Ltd,, UK) and a Parkinson-type leafchamber. The first 3 fully expanded leaves from each of3 plants from each of the 3 temperature treatments wererepeatedly measured 8 h after photoperiod initiation onthe same days on which nitrogenase and NR activitieshad been measured.

Detopping study

A study was conducted to determine the effect of shootremoval on nitrogenase and root NR activities. Plantswere acclimated in the growth chamber as describedabove, except that only the high nitrate fertihzer wasapplied in this experiment. Three plants were assayedfor nitrogenase and NR activities as described, butplants were repeatedly sampled for root NR activity byremoving a subsample of roots from the pot using a7-mm cork-borer. No nodules were present in the 5small sample cores removed from any plant. The holesleft by the removal of roots and sand substrate werefilled with silica sand immediately after the sampleswere taken. Intact plants were assayed for initial nitro-genase and NR activities immediately prior to shootexcision. The detopped root systems were then assayedfor nitrogenase and NR activities 1, 2, 3 and 6 daysfollowing removal of the shoots.

Results

Because the 3 temperature treatments had no apparentdifferential effects on the relative decline in root NR,leaf NR, nitrogenase activities or photosynthetic param-eters, data from al! temperature treatments were pooledfor statistical analyses.

Dinitrogen fixation activity

There were significant differences in nitrogenase activ-ities per plant prior to chilling between the fertilizertreatments, Nitrogenase activity averaged 27 jimol ethy-lene plant"' h"' for the low nitrate fertihzed plants and16 nmol ethylene plant"' h"' for those from the highnitrate fertilization regime. Prior to chilling, however,specific nodular nitrogenase activities of black alderseedhngs treated for 2 weeks with either nitrate fertil-izer were not statistically different, Nitrogenase activityof plants assigned to the low nitrate fertilization regimeaveraged 63 \imo\ ethylene (g nodule dry weight)"' h"'and piants assigned to the high nitrate fertilization re-gime averaged 54 |imol ethylene (g nodule dry weight)""'h"'. Within 1 day of chilling, nitrogenase activities de-clined by 33% for plants in the low nitrate treatmentand 41% for plants in the high nitrate treatment group,Nitrogenase activities of plants from either fertilizationregime had recovered to pre-chilling levels within 7 daysfollowing cold temperature treatment (Fig, 1),

Ftiysiot, Plant 82,1991 553

2 3 4 5 6 7 8DAY

Fig. 1, Specific nitrogenase activity of chilled black alder seed-lings receiving low nitrate (D) or high nitrate (•) fertilizerexpressed as timol ethylene produced (g nodule DW)"' h"'.Arrow indicates time chilling treatment was initiated. Verticalbars represent LSD at P = 0,05,

NR aetivity

Mean NR activities for leaves prior to chilhng were 2.8nmol nitrite (g fresh weight)"' h"' for alders receivinglow nitrate fertilizer and 1,6 jimol nitrite (g freshweight)"' h"' for alders in the high nitrate fertihzationregime. Although leaf NR activities increased in plantsfrom both fertilizer regimes the day after chilling, onlyin plants that had received the higher level of nitratefertilizer were leaf NR activities significantly increased(160% of pre-chilling leaf NR activity). Leaf NR activ-ities declined to pre-chilling rates by 7 days after coldtreatments were initiated (Fig, 2A),

NR activities of fine roots prior to chilling averaged3,0 nmol nitrite (g fresh weight)"' h"' for low nitratefertilized plants and 6,5 nmo! nitrite (g. fresh weight)"'h"' for high nitrate fertilized plants. Root NR activitiestended to increase after cold treatments similarly to leafNR activities. Root NR activities of plants receiving lownitrate fertilizer increased significantly to a mean of 3,7nmol nitrite (g fresh weight)"' h"' compared to pre-chilling activities. However, plants receiving high ni-trate fertilizer showed no significant change in root NRactivities (Fig, 2B),

PS activity

One day after chilling, net PS declined by 17 and 19%for low and high nitrate fertilized plants, respectively(Fig. 3A). Plants in the high nitrate fertilization grouphad greater mean net photosynthetic activities through-out the experiment compared to those of plants in thelow nitrate fertilization group, Stomatal conductanceand Cj responded differently to chilling depending on thenitrate fertilizer treatment (Fig, 3B, G). Plants receivinghigh nitrate showed a decline in g after chilling, whiielow nitrate fertilized plants showed no significantchange (Fig, 3B), The day after chilling, q decreased by

5% in high nitrate plants, but increased by 13% in lownitrate plants (Fig, 3C),

Oetopping study

Detopped plants showed a decline in NR and nitroge-nase activities after the shoots were removed (Fig. 4),Nitrogenase activity tended to decrease at a faster ratethan NR activity and was at < 5% of initial activity byday 4, while root NR did not exhibit a similar decline inactivity until day 7.

Discussion

There was significantly greater nitrogenase activity on awhole plant basis for alders receiving low nitrate fertil-izer for a 2-week period prior to chilling than for thosereceiving high nitrate fertilizer. Studies have showncombined N to inhibit nitrogenase activity of legumi-nous and actinorhizal nodules to various degrees, de-pending on N source and concentration, plant age andtiming of N addition (Cote and Dawson 1989, Granhallet al, 1983, Mackay et al, 1987, Streeter 1985a,b),

Reductions in nodule mass per plant have been ob-served in leguminous and actinorhizal plants receivinghigh levels of combined N (Granhall et al, 1983, Ro-berts et al, 1983, Streeter 1985a), although nodule dryweights between fertilizer treatments of our plants were

6 B2 3 4 5

DAYKg, Z, In vivo NR activity of A, leaves and B, iine roots, ofchilled black alder seedlings receiving low nitrate (D) or highnitrate (B) fertilizer expressed as nmol nitrite produced (gtissue FW)~' h"',- Arrow indicates time cMlling treatment wasinitiated. Vertical bars represent LSD at P = 0.05,

554 Ptiysiot, Plant. 82, t

Sitna>

c

oos:a.

^~'m

cu

CVl

oO

-—

15

14

13

12

11

10

g

go

0,5

0,4 -

0,3 -

0,2 •

0.1

2S0

o

rnal

Sc

—

8•g.

260

240

220

200

Fig, 3. A, photosynthesis, B, stomatal conductance and C,internal CO2 concentration of chilled black alder seedlingsreceiving low nitrate (•) or high nitrate (•) fertilizer. Arrowindicates time chilling treatment was initiated. Vertical barsrepresent LSD at P — 0.05.

not statistically different. The short, 2-week duration ofthe nitrate treatments probably precluded the detectionof any difference in nodule dry weig.ht accumulation dueto nitrate treatments in the present study. The lowervalues for specific nitrogenase activities ,of alders ex-posed to the higher nitrate treatment and measuredafter the 2-week-long nitrate fertilization treatment be-fore chilling were not significantly different from themean value for plants treated with the lower nitratenutrient solution. This was possibly due to the var-iability in nodule size among our 6- to 10-month-oldalders. Larger woody actinorhizal nodules tend to havelower specific nitrogenase activities than smaller nodu-les (Silver 1970).

The lack of significant differences in pra-chilling spe-cific nitrogenase activities between the 2-week-long ni-trate fertilization treatments agrees with work on nodu-lated leguminous black locust (Robinia pseudoacada

ivity

u

nto

o

100

SO

60

40

20

0

VV 11

\ \

1 2 3 4 5 6 7DAY

Fig. 4. Percent changes relative to day 1 measurements of rootNR activity (!•) and specific nitrogenase .activity (D) of de:topped black alder seedlings receiving high nitrate fertilizer.Shoots were removed from plants shortly after measurementson day 1 (arrow). Vertical bars represent LSD at P = 0,05,

L,) that showed that long-term fertihzation with com-bined N at a concentration similar to the high nitratefertilizer used in our study had no effect on specificnitrogenase activity compared to that of plants receivingno combined N (Roberts et al, 1983),

Nitrogenase activity of actinorhiz.al nodules seems todepend upon current photosynthate, when available,rather than upon stored carbohydrates (Wheeler 1971).Nitrogenase activities show a diurnal rhythm thatclosely follows photosynthetic activity, and plant leafarea is closely correlated with whole plant nitrogenaseactivity (Dawson and Gordon 1979, Huss-Danell andSellstedt 1985), Chilling the shoots of some plants caninhibit carbon translocation (Potvin 1988) and chillingthe shoots of black alder seedlings in our experimentreduced net PS by 17 to 19% (Fig. 3A), which wouldnormally result in decreased nodular nitrogenase activ-ity (Gordon and Wheeler 1978), Results from the de-topping experiment further illustrate that nitrogenaseactivity is dependent upon the shoot, because activitydropped considerably 1 day after shoot removal (Fig,4), In our experiments, black alder seedlings of thesame age and exposed to the same treatments as plantsassayed for nitrogenase and NR activities showed lowerproportional decreases in net PS (Fig, 3A) than in nitro-genase activities (Fig, 1), which suggests that mecha-nisms other than a decline in PS were partly responsiblefor the decreases in nitrogenase activities after chilling.

Decreases in nitrogenase activities with cold temper-atures have been well documented for actinorhizal spe-cies (Cote .and Dawson 1989, Huss-Danell et al, 1987,Waughman 1977), Complete cessation of nitrogenaseactivity usually occurs at nodule temperatures of 5 to8°C, with recovery of activity within hours of a return topre-chilling temperatures (Huss-Danell et al, 1987,Winship and Tjepkema 1985). Loss of nitrogenase activ-ity due to chilling of alder nodules seemed to be de-pendent upon a temperature-sensitive gas-diffusion bar-

Ptiysiol. Ptant. 82, t99t 555

rier (Winship and Tjepkema 1985), while decreasedlevels of active nitrogenase explained some loss of activ-ity as well (Huss-Danell et al, 1987). In the presentstudy entire plants were subjected to chilMng temper-atures for a duration of 2.5 h, while in other studiesroots or excised nodules were chilled for a period ofapproximately 1 h. The relatively long duration of coldtreatment in our study may have caused more extensivedamage or metabolic imbalance in the nodules thatwould require increased time for recovery. The chillingof alder nodules (Winship and Tjepkema 1985) and theexposure of white clover nodules to high nitrate concen-trations (Minchin et al, 1986a) both increased the resist-ance of the nodule oxygen diffusion barrier. A possiblesynergistic increase in resistance to oxygen diffusion inthe nodule due to both chiOing and expostire to thehigher nitrate concentration in the current study mayhave resulted in the proportionally greater inhibition ofspecific nitrogenase activity of alders in the higher ni-trate treatment after chilling,

Photosynthetic activity correlates with plant N nutri-tion because chlorophyll and the enzymes responsiblefor photosynthesis and metabolistn require N (Kramerand Kozlowski 1979), This seemed to be true when thephotosynthetic rates of alders receiving high or lownitrate fertilizer were compared (Fig, 3A), The plantsreceiving high nitrate fertilizer had higher levels of PSand darker green leaves than the low nitrate fertilizedplants, suggesting that chlorophyll concentration wasgreater in the leaves of the high nitrate fertilized plants.

There were differences in the chilling responses of qand gj between nitrate fertilizer treatments (Fig, 3B, C),Alders receiving high nitrate fertilizer exhibited a 39%decline in g,, concurrent with a decline of only 5% in c,.This suggests that the 17% decrease in PS was not duemainly to stomatal limitation of COj diffusion. The lownitrate fertilized plants showed no significant change ingj after chilling, but did show an increase in q, whichargues for increased mesophyll resistance as an impor-tant factor in the reduction of PS in these plants (Far-quhar and Sharkey 1982), The chiUing treatments in thecurrent study probably caused biochemical lesions da-maging to the photosynthetic process of plants in bothfertilization regimes,

Neave et al, (1989) found that field grown, matureblack alders were able to partially recover from reducedPS within a few days after a night frost during autumn.The ctirrent study showed that black alder seedlingsrecovered their pre-chilling rates of PS less well than didblack alder in the field. Our seedlings were growitigvigorously and had not been exposed to reduced day-length and cooler temperatures to induce "cold harden-ing" (Oquist 1983) of the plants, suggesting the possibil-ity of some permanent damage due to the chilling treat-ments.

Root NR activity dechned after removal of the shootat a lesser rate than did nitrogenase activity and tooklonger to reach rates 5% of those prior to detopping

(Fig, 4). It is possible that nodular nitrogenase activity ismore dependent on current photosynthate than areother energy-requiring root .activities (Walsh et al, 1987)or that root NR activity is a stronger sink for carbo-hydrates mobilized from root tissue than is nodularnitrogenase. It is also possible that detopping increasedthe resistance of the nodule oxygen diffusion barrierdue to reduced photosynthate supply to the nodules(Minchin et al, 1986b),

The level of NR in higher plant tissues is usuallyassoci.ated with nitrate supply to the tissue (Guerrero etal, 1981) and this pattern was apparent for root tissuesin the present study (Fig, 2B). Analyses of leaf nitrateconcentrations showed that leaves from each of thenitrate treatments had similar nitrate concentrations of< 5 nmol nitrate (g tissue dry weight)"' (data notshown), Pizelle and Thiery (1986) found no evidence ofnitrate in the transpiration stream of field-grown aldersand no correlation between soil nitrate concentrationsand leaf NR activity. Black alders growing nonsymbiot-ically and receiving combined N in the form of nitrate,ammonium or nitrate plus ammonium exhibited negli-gible leaf NR activity compared to alder growing sym-biotically and receiving no combined N (Blacquiere andTroelstra 1986). The higher activity in ieaves of symbiot-ically grown alder was not of phylloplane microbialorigin, and a significant positive correlation betweengrowth and leaf NR activity of nodulated black alderreceiving no combined N has been observed (Benamaret al. 1989), Alnus incana was shown in an in vitro NRassay to have roots and shoot tips that were responsiveto soil nitrate concentrations, although leaves exhibitedno activity regardless of the soil nitrate status (Sellstedt1986), Another dinitrogen fixing tree, leguminous blacklocust, was shown to possess constitutive leaf NR activ-ity that decreased over time as nitrogenase activity be-gan to increase during spring and summer (Boutekrabtand Pizelle 1989), Black alder leaves show NR activityunrelated to the level of substrate or leaf nitrate. LeafNR activity of our black alder seedlings and those ofBlacquiere and Troelstra (1986) corresponded moreclosely to nitrogenase activities in the root nodules. Liand Gresshoff (1990) found a constitutive NR enzymein soybean leaves that responded to a signal moleculefrom nodules, possibly allantoic acid, and they sug-gested that this constitutive NR may be primarily in-volved in ureide metabolism rather than in in vivo ni-trate metabolism.

Cote et al, (1989) observed that black alder seedlingsreceiving all of their N from combined sources in thesoil had no net resorption of leaf N in the autumn,whereas net resorption of leaf N increased with a plant'sdependence on fixation of atmospheric Nj, Our resultssuggest that autumnal cooling would decrease nodulardinitrogen fixation more than it would decrease rootNR activity. Thus the amount of N available to movefrom roots to leaves of black alder under cool conditionswould be greater in plants proportionally more depend-

556 Ptiysiot. Ptant. 82, IWl

ent on nitrate than on dinitrogen fixation. If the trans-location rate of N from leaves during the autumn or acool period remained similar for all plants, then blackalders more dependent on nitrate than nitrogenase ac-tivity for their N nutrition would exhibit less net resorp-tion of leaf N,

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