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Effects of salinity andnitrogen source on growthand nitrogen fixation inalfalfaRachid Serraj a & Jean‐Jacques Drevon b

a Département de Biologie, Faculté desScience‐Semlalia , BP S.15, Marrakech, Moroccob Laboratoire de Recherche sur lesSymbiotes des Racines , INRA , place Viala,Montpellier‐Cedex, 34060, FrancePublished online: 21 Nov 2008.

To cite this article: Rachid Serraj & Jean‐Jacques Drevon (1998) Effects ofsalinity and nitrogen source on growth and nitrogen fixation in alfalfa, Journal ofPlant Nutrition, 21:9, 1805-1818, DOI: 10.1080/01904169809365525

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JOURNAL OF PLANT NUTRITION, 21(9), 1805-1818 (1998)

Effects of Salinity and Nitrogen Source onGrowth and Nitrogen Fixation in Alfalfa

Rachid Serraja and Jean-Jacques Drevonb,1

aDépartement de Biologie, Faculté des Science-Semlalia BP S.15, Marrakech,MoroccobLaboratoire de Recherche sur les Symbiotes des Racines, INRA, place Viala,34060, Montpellier-Cedex, France

ABSTRACT

The Interaction between the effects of nitrate (NO3) and sodium chloride (NaCl)concentration on growth) water relations, nitrogen (N) contents and N fixationwere investigated in alfalfa (Medicago sativa L. cv. Magali). The plants weregrown hydroponically in a growth chamber, in the presence or absence of 3mM potassium nitrate (KNO3) and exposed to various concentrations of NaCl.Increased salinity resulted in a significant decrease in shoot and root biomass,relative water content and water potential. Shoot growth was more inhibitedby NaCl than root biomass. The plants grown in the presence of NO3 wereslightly less affected by NaCl than the plants dependent on N fixation for theirN nutrition. Nitrogenase activity measured by acetylene reduction activitywas substantially inhibited by NaCl, and this inhibition was significantlycorrelated to the inhibition of shoot growth and total N contents. Thecomparison of the curves of ARA response to oxygen (O2) partial pressureshowed that the salt-induced inhibition of nitrogenase activity was associated

1Corresponding author.

1805

Copyright © 1998 by Marcel Dekker, Inc. www.dekker.com

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1806 SERRAJ AND DREVON

with a significant increase in the critical O2 pressure of the nodules exposed toNaCl. This result shows that NaCl decreases the nodule permeability to O2

diffusion in undeterminate nodule of alfalfa, like previously shown withdeterminate nodules of soybean.

INTRODUCTION

Alfalfa is one of the major crops in the arid and semi-arid regions especially inthe mediterranean basin, where symbiotic N2 fixation contributes to its adaptation.Unfortunately, soil salinity is also common in arid and semi-arid regions (Lauchliand Wieneke, 1979) and N2 fixation can be extremely sensitive to salinity stress(Bernstein and Ogata, 1966;Bekkietal., 1987; Serraj etal., 1994).

Most legume species have been found to be either sensitive or only moderatelysalt tolerant (Bernstein and Ogata, 1966; Delgado et al., 1994) and the necessity todevelop salt tolerant symbiosis has long been emphasized (Singleton et al., 1982;Epstein, 1985; Sprent and Zahran, 1988). Although the success of such an approachwould require the improvement of both partners of the symbiosis, it is generallyconcluded that rhizobia are relatively more salt tolerant than the correspondinghost legumes (Singleton et al., 1982). Nevertheless, considerable variability in salttolerance for plant growth has been reported among and within legume species(Pessarakli and Zhou, 1990; Delgado etal., 1994).

Legumes can assimilate simultaneously both N2 and mineral N, and an interactionbetween N nutrition source and salinity has been reported (Cordovilla et al., 1995).Several reports showed thatN2-fixing legume plants were more sensitive to salinitythan those dependent only on mineral N (Tu, 1981; Singleton and Bohlool, 1983).A beneficial effect of N fertilizers on growth under saline conditions has beenreported in non-legumes (Lewis et al., 1989). However, the interaction betweensalinity, N fertilization and N2 fixation is further complicated by the inhibitoryeffects of mineral N on nodule growth and function (Heckmann et al., 1989; Serrajetal., 1992).

Although salinity effects on N2 fixation have been extensively studied in severallegume species, the mechanisms of such inhibition are still poorly understood.Sprent and Zahran (1988) reported that salinity inhibits the expansion and curlingof root-hairs and reduces the number of nodules in faba bean. According to Bekkiet al. (1987), Hafeez et al. (1988), and Singleton and Bohlool (1983), nodule activityappears less affected by salt stress than plant growth and nodulation. However,there is little information on the short-term response of nodule nitrogenase activityto salt stress. Sprent (1972) observed a rapid decrease in the acetylene reductionactivity of excised soybean nodules exposed to NaCl, even though excised nodulesmay already have reduced activity. Serraj et al. (1994) showed that exposure ofintact soybean plants to 0.1 M NaCl resulted in a rapid decrease in nitrogenaseactivity and nodule respiration, indicating an effect of salt stress on the permeability

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EFFECTS OF SALINITY AND N SOURCE ON ALFALFA 1807

to O2 diffusion. It is also well established that an O2 limitation is associated withnitrogenase activity inhibition by various other environmental limitations includingNO3 application (Vessey et al., 1988), drought (Serraj and Sinclair, 1996) and phloemsap deprivation (Hartwig et al., 1987). Several hypotheses of nodule permeabilityto O2 regulation in the nodule cortex by environmental factors have been recentlyproposed, involving glycoproteins (Iannetta et al., 1995), osmoelectric regulation(Denison and Kinraide, 1995) and osmocontractil cells in the inner nodule cortex(Drevonetal., 1995).

In this work, our first objective was to compare the effects of NaCl concentrationon growth, water relations and N accumulation in the presence or absence ofmineral N. Secondly, we investigated the responses of nitrogenase activity toraising external oxygen pressure in order to test whether intranodular O2 diffusionis involved in the inhibition of nitrogenase activity by NaCl in alfalfa undeterminatenodules like previously observed in determinate soybean nodules.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Seeds of alfalfa {Medicago sativa L. cv. Magali) were surface-sterilized in 6%(w/v) calcium hypochlorite for 15 min, washed thoroughly with sterile water, andgerminated at 28°C in moist autoclaved perlite for 48 h. Seedlings were inoculatedwith a liquid inoculant of Rhizobium meliloti strain 2011 (10s bacteria mL"1) andtransferred for growth into a 0.5 L container. The culture containers were two-thirdsfilled with a minus-N hydroponic nutrient solution (Kalia and Drevon, 1985) andarranged randomly in a growth chamber. The nutrient solution was renewed daily,the pH was maintained close to 7.0 by adding 0.2 g L1 calcium carbonate (CaCO3)and air was continuously bubbled through the solution at a flow rate of 0.4 L min1.During the 16 h photoperiod, the irradiance, supplied by mercury vapor lamps(OSRAM HQI-T400 W/DH), was about 500 umol nv2 s1. Day/night temperatureswere22°C/20°C.

During the first 20 days, i.e., before nodule emergence, the nutrient solution wascomplemented with 2 mM urea. After this period, the plants were transferred to anew nutrient solution either without N or with 3 mM potassium nitrate (KNO3), aconcentration that inhibits completely nodulation (Serraj et al., 1992). At thisstage, the plants were exposed to salinity by adding NaCl to the growth medium(final concentration 0,25,50, or 100 mM).

Nodule Gas-Exchange Measurement

Before nodule gas-exchange measurement, the nodulated-root compartment wassealed and flushed with a humidified air-flow at a rate of 0.1 L min1. The volume ofnutrient solution was maintained at 10% of the volume of this compartment so thatthe root nodule mass was above the nutrient solution.

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1808 SERRAJ AND DREVON

The acetylene reduction activity (ARA) and its response to pO2 were assayedafter three weeks of treatment in an open-flow system described in detail by Drevonet al. (1988), Outflow ethylene concentration was determined with a gaschromatograph equipped with a flame ionization detector. ARA response to O2

enrichment was measured using the same system, on three plants per treatment(control or salt-treated), by varying the O2 and N2 partial pressures as describedpreviously by Heckmann et al. (1989). The mass flowmeters (Tylan, Z.I. ChesnesLuzais, La Verpillere, France) were used to vary the composition of the gas mixtureand maintain a 0.1 L min1 constant flow during the assay. To prevent inhibition ofnitrogenase activity by O2 excess, the increase in pO2 was imposed graduallyrather than by a step change. A concomitant increase in diffusion barrier resistanceduring pO2 alteration may prevent the inhibition of nitrogenase activity by O2

excess. ARA was measured at 5, 10,20,30,40,50, and 55 kPa O2. Plants wereexposed to each pO2 for 20 min to reach and maintain the new steady state. Theoutflow C2H4 concentration was determined at least three times during the steadystate. Approximately 2.5 h were required for the complete measurement of ARAresponse to pO2. After the measurements, nodule dry weight was determined.

Plant Harvest and Analysis

Plant were harvested three weeks after treatment initiation with six replicates pertreatment. Roots and nodules were separated from the shoots, rinsed, and driedwith filter paper. All plant parts were immediately weighed for FW determinations.Dry weight (DW) was measured after drying for 48 h at 75°C. Leaf water content(WC) was calculated as the difference between FW and DW in % FW. Leaf waterpotential was determined at midday on the first fully developed leaf using a pressurechamber (Scholander et al., 1965). Total N content was analyzed according to themethod described by Bremner (1965).

Anova tests were used to analyze the data with SigmaStat Software. Statisticsof regressions were computed as described in Godfrey and Drevon (1991) withcovariance analysis and differences between estimates were analyzed withStudent's t-tests.

RESULTS AND DISCUSSION

Plant Growth

Without NaCl, NO3-fed plants had significantly higher DW accumulation intheir shoots and roots than N2-fixing plants, although the shoot/root ratio washigher in the later (Figure 1). Root growth was more inhibited by NaCl for NO3-fedplants than for N2-fixing plants. Moreover, the inhibitory effect of NaCl on rootgrowth was most pronounced under 25 mM NaCl. Indeed, there was no significantdifference in root growth under 25,50, and 100 mM NaCl whatever the N source.

For both N sources the inhibitory effect of NaCl on shoot growth was similar(Figure 1). Shoot growth was more inhibited than root growth by higher NaCl

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EFFECTS OF SALINITY AND N SOURCE ON ALFALFA 1809

O - N:y= 0.98 - 0.01x + O.OOOlx2, ra= 0.98

N: y= 2.08 - 0.02x + O.OOOlx2, r '= 0.872O - N: y= 1.53 - 0.01x + 0.0001x2, r '= 0.82

25 50 75 100

NaCl (mM)

FIGURE 1. Effect of NaCl concentration on alfalfa shoot (A) and root (B) growth in thepresence (+N) or absence (-N) of 3 mM KNO3. Each value is the mean (±SE) of 6 replicates.

applications. The shoot/root ratio was decreased by NaCl in N2-fixing plants (from0.95 in control to 0.31 under 100 mM NaCl) although it maintained in NO3-fedplants.

These results confirm the previous reports on soybean and alfalfa (Bernsteinand Ogata, 1966; Khan et al., 1994), chickpea (Lauter et al., 1981), and faba bean(Yousef and Sprent, 1983) grown in the field or in the greenhouse showing thatshoot growth was more inhibited by NaCl than root growth, regardless of the Nsource.

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1810 SERRAJ AND DREVON

80

78

155

74

72

-0.4

-0.6

-0.8

To -1.0

-1.6

-1.8

-2.0

i • • • • i i • • • • i

• +N: y= 77.73 -0.101X + 0.0006X2, r»= 0.99O -N: y= 78.04 - 0.107x + 0.0006x2, r '= 0.98

• +N: y= -0.55 -0.017X + 0.0005X2, r'= 0.94l O pN: y= -0,62 : O.qi^x + O.q0p7x', r '= p.96

25 50 75

NaCl (mM)100

FIGURE 2. Effect of NaCl concentration on alfalfa leaf water content (A) and waterpotential (B) in the presence (+N) or absence (-N) of 3 mM KN03. Each value is the mean(±SE) of 6 replicates.

Water and Nitrogen Contents

Leaf water potential and water content were both significantly decreased byNaCl (Figure 2), without any significant effect of N source on these parameters.The lack of interaction between NO, and NaCl on leaf water potential and watercontent has not been described previously, to our knowledge. One possibleinterpretation of this result is that NO3 and NaCl interaction could affect the ionicrather than the osmotic components of salt stress. This interpretation contrastswith the conclusion of Munns (1993) suggesting a biphasic model to explain plant

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EFFECTS OF SALINITY AND N SOURCE ON ALFALFA 1811

45

~ 40Ia

n

Q .

aE

nte

oo

3oH

35

30

25

20

15

10

5

3.0

~ 2.5

IoO 2.0Z

1.5

A i

• +N: y = 38.87 - 0.50 x + 0.003 x2, r '=0.99O -N: y = 28.65 - 0.30 X + 0.001 x2, r »=0.99

> +N: y = 2.94 +0.01 x-0.0001 x2, ̂ =0.91O ^N: y = 2.60-0.02 x + 0.000^ x2, r

25 50 75

NaCl (mM)

100

FIGURE 3. Effect of NaCl concentration on alfalfa total N contents in mg per plant (A)and in % DW (B) in the presence (+N) or absence (-N) of 3 mM KN03. Each value is themean (±SE) of 6 replicates.

growth response to salinity, the first effect of salinity would be osmotic, followedby a toxic effect linked to Na+ and/or Cl' accumulation.

Without NaCl, NO3-fed plants had significantly higher N accumulation in shootsthan N2-fixing plants (Figure 3). Nitrogen accumulation was more inhibited byNaCl for NO3-fed plants than for N2-fixing plants, except under 100 mM NaCl.Con-sequently, the N-use efficiency (NUE=mg D W/mg N) was increased by NaCltreatment in the NO3-fed plants (from 38 in control to 67 under 100 mM NaCl)

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1812 SERRAJ AND DREVON

5aa>3

•ooz

r—

!cI *

o""5E

|

85

80

75

70

65

60

55

50

160

150

140

130

120

110

100

90

80

- ' • i • • • * i • • • • 1 • • ' • 1 • ' ' ' 1 ' ' • -

: A :

- P\ ~'• -L >. _ :

7 1 -

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7 y= 77.13-0.299X + 0.002X2, r2=0.93 -.

• I B̂

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p-

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T V '•— *̂~̂ _̂_̂ I -_- y = 1 5 7 . 2 5 - 1 . 4 3 7 x + 0 . 0 0 7 X 2 , r • = 0 . 9 9 | ^

1 , . . . 1 1 , , . :

25 50 75

NaCl (mM)

100

FIGURE 4. Effect of NaCl concentration on alfalfa nodule mass (A) and acetylenereduction activity (ARA) (B). Each value is the mean (±SE) of 6 replicates.

although it maintained at about 35 in N2-fixing plants. This corresponded to adecrease in N concentration (N content in % of plant biomass) of the NO3-fedplants under 100 mM NaCl although this concentration was not affected by milderNaCl treatments. By contrast the N concentration of N2-fixing plants was mostlyaffected by 25 and 50 mM NaCl.

The absence of a significant effect of NaCl on percent N content of NO3-fedplants (Figure 3B) supports the previous reports showing inhibitory effects ofNaCl on growth, without any decrease of N percent (Pessarakli and Zhou, 1990;Cordovilla et al., 1995) although the interaction of salt and NO3 concentrations may

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EFFECTS OF SALINITY AND N SOURCE ON ALFALFA 1813

2.0 -

5 10 15 20

ARA (umol C2H4 h"1 plant"1)

FIGURE 5. Plot of shoot DW (A) and total N content (B) as a function of acetylenereduction activity for alfalfa plants exposed to various NaCl concentrations.

depend upon growth conditions and rates (Sprent andZahran, 1988). This contrastswith the relatively high effect of NaCl on the percent N content of N2-fixing plants(Figure 3B), despite there being only a slight difference in total N content of shootsbetween both N treatments (Figure 3 A). Therefore, N accumulation appears to bemore salt sensitive in N2-fixing plants than in NO3-fed plants.

Nodule Nitrogenase Activity

Nitrogenase activity was not detectable in NO3-fed plants for which nodulationand nodule function were inhibited by the weekly 3 mM NO3 supply. For the

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1814 SERRAJ AND DREVON

N2-fixing plants which received urea only during the first three weeks withoutNaCl, the inhibition of nodulated-root C2H2 reducing activity (ARA) was almostproportional to the concentration of NaCl in the nutrient solution and was higherthan that on nodulation (Figure 4). The above response of ARA to NaCl wassimilar to that of shoot/nodule ratio calculated from values in Figure 1A and 4A.

Nodule ARA was significantly correlated with both shoot DW and total Ncontents when data from all NaCl treatments were combined (Figure 5). Thecorrelation between total N contents and nodule ARA for the N2-fixing plants(Figure 5) indicates that the inhibition of nitrogenase activity by NaCl, as measuredby ARA assay, corresponded to a substantial reduction of N fixation, andconsequently plant growth (Figure 1).

The inhibition of N fixation with NaCl treatment was mainly due to a reduction ofnitrogenase activity per g nodule, while nodule growth was only slightly decreased.This agrees with previous observations in faba bean by Sprent and Zahran (1988)who concluded that nodule growth compensated for inhibitory effect of NaCl onnodule activity. This indicates that the effects of salinity on nodule developmentand growth may not always be the major processes determining legume responseto this stress (Singleton and Bohlool, 1983; Hafeez et al , 1988). Thus, specificnitrogenase activity could be a key factor in the response of N2-fixing legumes tosalinity. This is further substantiated in our work by the higher critical O2 pressure(COP) of nodules grown with NaCl.

The responses of nodule ARA to rhizosphere pO2 were significantly differentamong NaCl concentrations (Figure 6). The COP, inferred from these curves as theoptimal pO2 for ARA was close to 40 kPa O2 for the control plants (0 mM NaCl)whereas it was above 55 kPaO2 for the plants exposed to 50 mM or 100 mM NaCl.Thus, NaCl diminished the effects of increasing external pO2 on nodule ARA.Importantly, the inhibition of nodule ARA by 50 mM NaCl was completely reversibleby increasing pO2 around the nodules which indicated that an O2 limitation withinthe nodules may have inhibited respiration and nitrogenase activity. Contrastinglyfor nodules exposed to 100 mM NaCl, ARA stimulation by pO2 did not result in anyrecovery from salt inhibition which suggested that nitrogenase in this stage isconstrained by factors other than pO2. A similar conclusion has been made withsoybean nodules exposed to various levels of severity of drought stress (Diaz delCastillo et al., 1994; Serraj and Sinclair, 1996).

The increase in the COP (critical O2 pressure, i.e., pO2 above which nodulemetabolism was no more stimulated by raising pO2) observed in the salt-treatednodules (Figure 6) is in agreement with our previous data on soybean (Serraj et al.,1994) and indicates a decrease in nodule permeability to O2 diffusion. Thisinterpretation is consistent with the model of nodule O2 regulation proposed byDrevon et al. (1995), and theNaCl-linked contraction of cells in the nodular innercortex (Serraj et al., 1995). Interestingly, ARA responses to rhizosphere pO2 afterthree weeks with NaCl were comparable to those observed after a few hours withNaCl which suggests that in the presence of salt the nodule cannot adapt its

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EFFECTS OF SALINITY AND N SOURCE ON ALFALFA 1815

50

•^ 40a"5.

^ 30

o

rf 10

0 -

I I • I I

Control: y=-0.32 + 2.21x-0.03x2, r*= 0.98© 50mMNaCI: y=-0.05 + 0.78x - 0.0O4X2, r'= 0.99O 100,mM NaCl,: y= 1.53 f-0.07x, r'p 0.99. ,

10 20 30 40

pO2(kPa)

50

FIGURE 6. Effect of NaCl concentration and external pO2 on acetylene reduction activityof alfalfa nodules. Each value is the mean (±SE) of 3 replicates.

permeability to overcome the O2 deficiency that we have previously shown to beinduced by NaCl treatment (Serraj et al., 1994).

CONCLUSIONS

The main conclusions of this work are that (i) N2-fixing alfalfa plants are moresalt sensitive than NO3-fed plants and (ii) the nodule nitrogenase activity, and itsregulation by O2 could be key factors in the response of N2-fixing plants to salinitystress.

The effects of NaCl on plant growth showed a higher degree of inhibition in theshoot than in root, whatever the N treatment, which was in agreement with severalreports that have previously shown that shoots were more affected by salinitythan roots. Our results showed a higher effect of NaCl on the percent N content ofN2-fixing plants, compared to NO3-fed plants which indicated that N2 fixation wasmore sensitive to NaCl than NO3 nutrition or the other functions supporting plantgrowth.

The inhibition of N2 fixation by NaCl treatment over the three weeks ofexperimental period was mainly due to a reduction of nitrogenase activity per g

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1816 SERRAJ AND DREVON

nodule, whereas nodule growth was only slightly decreased. This result is inagreement with the previous work on the short-term effects of salt stress on N2

fixation in soybean (Serraj et al, 1994), and indicate an important role of the nodulenitrogenase activity in the response of N2-fixing plants to salinity. This conclusionis further supported by the higher critical O2 pressure of nodules grown with NaCl.

The interaction of salt stress and pO2 on nitrogenase activity depends upon theconcentration of salt. The inhibition of nitrogenase activity by moderate saltstress (50 mM NaCl) was completely reversible by increasing pO2, whereas a moresevere stress (100 mM NaCl) resulted in an inhibition that was not reversed in therange of pO2 tested. An important future approach in studying the N2 fixationresponse to salt stress may be a comparison of physiological performance amonglegume species and cultivars and to examine differences in the physiological traitsassociated with nodule establishment and activity under salinity conditions.

ACKNOWLEDGMENTS

This work was supported by an European Community grant (CEE 02 087/TSDA-180). The postdoctoral journey of R. Serraj inlNRA-Montpellierwas supportedbyanAUPELFgrant .

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