Indian Journal of Experimental Biology Vol. 37, September 1999, pp. 843-848

Review Article

Effect of salt stress on nodulation and nitrogen fixation in legumes

K Swaraj Department of Botany, CCS Haryana Agricultural University, Hisar 125 004, India

and

N R Bishnoi*

Department of Environmental Science and Engineering, Guru Jambheshwar University, Hi sar 125 00 I. Indi a

It is now well established that almost all phases of root nodule development in legumes are adversely affected by saline conditions in the rooting mediu m. There is also a general agreement that the rhi zobia are more tolerant to salt stress than the host plant, but they show considerable strai n variability in growth and survival under saline conditions. Inhibitory effect of salinity on nodu lation has been attributed to decrease in rhizobial coloni sation and shrinkage and lack of root hair form a­tion. Salt stress also induces premature senescence of already formed nodules. Both NT fix ati on activi ty and nodul e respira­tion are inhibited sharply on exposure of plants to saline conditions. The decrease in NT fix ation has been ascribed to direct effect on nit rogenase acti vity or an indirect effect through decrease in leghemoglobin content , respiratory rate. malate con­centrations in nodules and photosynthate availability. Salinity increases oxygen diffusion resistance in the nodules and alters their ultrastructure. Decrease in NT fixation in nodules under salinity is also accompanied by. parallel decrease in the activity of H20 r scavenging enzymes li ke catalase, ascorbate peroxidase and the level of ant ioxidants li ke ascorbic ac id . Nodul es appear to undergo osmoregul ati on under saline conditions by accumulating physiologically compatibl e solutes like proline. sugars (pinnitol) and lacti c acid. The intensity of the adyerse effects of salinity on nodule functioning depends on plant spe­cies, rhizobial strai n, duration of exposure to saline conditions, nature, concentration and mode of sa lt application.

Large tracts of cultivated land in the arid and semi­arid tropics are gradually getting infested with high soil salinity because of irrigation with poor quality water, high evaporation demands and application of chemical fertilizers. Salt stress imposes a major con­straint on productivity of legumes especially when plants are solely dependent for their N-requirement on symbiotic N2-fixation H

. In legumes salt stress can limit plant growth and available photosynthates4

,5,

reduce nodulation6 and decrease nodule Nr fixation and respiration7

•8

. The nodule functioning is adversely affected even by mild stress conditions which have no adverse effect on growth of plants dependent on com­bined nitrogen . Salt effects can be osmotic or nonos­motic (ionic toxicity and imbalance) in nature9

•1O

•

In the past few years, a lot of new scientific infor­mation on the effect of salinity on nodulation and Nr fixation has appeared in the literature. In view of the importance of the subject in sustainable agricultur~, it is prudent to critically assess in this review the re­sponse of the symbiotic system to soil salinity.

*Correspondent author Abbreviations: ARA='acetylene reduction activity. AOS=aciivated oxygen species, Lb=leghemoglobin . Nrase=nitrogenase, SOD= superoxide dis mutase.

Rhizobial microsymbiont and saline conditions

There is a general agreement ·that rhi zobi a are ab le to cope with salinity more than the host legume, but they show considerable stra in vari ability in growth and survival under saline conditions. Rhi zobia in symbiotic state live in cell s having approximately 300-400 mol m-), a solute concentrati on much higher than these concentrations faced by these organi sms in the free living conditions in the soi li l. Thus, in saline soils rhizobia mainly come across ionic stress. How­ever, rhizobia can grow under saline conditions by making ionic adjustments. Rhizobium sp WR I 00 I isolated from extremely drought and salinity tolerant legume Prosopisl 2 has been shown to undergo os­motic adjustment by enhanced glutamate produc­tion l). Recently Kohl et al.14 emphas ized the impor­tance of proline catabolism in bacteroid metabolism during environmental stress in soybean nodules.

Once inside the host, rhizobia may be subjected to low osmotic potential , but they are not like ly to face particles with a net e lectrical charge as they are usu­ally pumped into vacuoles even in halophytes and the cytoplasmic compartment-harbouring the endophyte adjusts osmotically by producing enzyme compatible solutes such as beta ines l5. Thus onl y in ex ternal envi-

844 INDIAN J EXP BIOL, SEPTEMBER 1999

ronment rhizobia encounter high inorganic salt con­centrations. There is lot of evidence indicating that certain ions such as HC03- are more toxic than others, but there is insufficient work separating pH and ionic effects to draw any definite conclusion .

Nodulation and N2 fixation The process of nodulation has been reported to be

highly sensitive to saline conditions3,6, 16, 17. Inhibitory

effect of salinity in soyabean has been attributed by some of these workers to decreased rhizobia l coloni-. zation and shrinkage and lack of root hair formation . However, symbiosis of soybean with salt to lerant Brady rhizobium results in lower salinity related inhi­bition of N2-fixation 18.

Although the effects of salinity on legume growth , nodulati on and N2-fixati on have been stud ied by a

b f k ] 19-21 .. h' . num er 0 wor ers" " yet companson Wit 111 van-ous species or symbi oses are complicated by applica­tion of salts at different concentration, by different modes, for different duration of times, at different stages of plant growth and measurement of ni troge­nase activi ty using conventi onal acety lene reduction assay. Thus soybean has recentl y been reported to be relative ly sa lt to lerant with 35 % decrease in C2H2reduction activity (ARA) following treatment with 100 mM NaCI by adding the salt to growth l!1e­dium through a progress ion of 25, 50 and 100 mM NaCI at 4 days interval for 28-30 days of growth8 and very sensit ive to sa lt with a 90% decrease in ARA following 24 hr exposure of hydroponicall y grown 30-40 days old plants to 100 mM NaC124. This dis­parity may re fl ect different modes of salt app licat ion and could in part be due to genotypic d ifferences wi thin thi s species25

. Among the 16 soybean cu ltivars (raised in sterile plastic jar assemblies, 500 ml each) screened for sa lt tolerance (80 mM NaCI) in re lation to symbiot ic Nrfixat ion, II have been found to be salt sensitive and the other five were moderate ly tol­erant with moderate growth, nodu le forma ti on and

Nr fixation 25.

The intensi ty of adverse effects of salinity on nod­ule development and Nrfixation also depends on growth conditions. Under low irradiance, these proc­esses in soybean were more sensiti ve to sa lt stress as compared to those under hi gh irradiance

25. '

Different legume species, differ in their relative re­sistance to salt stress. Although salt stress signifi­cantly reduced nodular dry we ight and ARA in all the four symbioses namely pea, faba-bean, common bean

and soybean, inhibition of ARA was more pro­nounced in pea and faba-bean and less pronounced in soybean8

. Common bean nodules show very little re­sponse to 50 mM NaCI, however, the higher concen­tration 100 mM NaCI leads to cons iderable reduct ion in ARA. The decrease in ARA in nodules of P. sati­

vum, P. vulgaris, G. max and V. faba under sa lt stress reflected itself in corresponding decrease in reduced N-content of plants. In P. sativum, the nitrogen con­tent of plants decreased from 267 mg/plant in contro l to 102 mg/plant under 100 mM NaCI treatment.

Among forage crops, alfalfa (Medicago sati va L.) responds to salt treatment (0 .15 M NaCI, 2 weeks) by 50% decrease in nodule number, reducti on in nodul e size and 30% decrease in A RA21 . In white lupin (LL/ ­pinus albus L.), which is relat ive ly more sa lt tolerant , growth parameters like nod ule number. vo lume, shoot and root dry weight, shoot photosy nthes is and respi­ration rate remain unaffected by 6 day exposure to NaCI concentrat ions26 up to 1':-0 mol m-'. Howe ver, increasi ng salt stress up to 100 mol m-J of NaCI leads to decrease in both total and specific nitrogenase ac­tivi ty27 , Clusterbean rai sed in sand cu lture conditions also appeared to be moderately sens itive to saline cond iti ons wi th ARA decreasing by 18-38% on treatment of plants wi th 50 mM NaCI for 3-6 days2x.

Among pu lses, chi ckpea (Cicer a riel /Ill/III L.) is hi ghly sensitive to sal ine conditi ons . Seven day long treatment of seven week o ld plants ra ised in sand ~u l ture and treated w ith 50 and 100 mol m-:1 NaCI resulted in 63-70% decrease in ARA'x. Surprisingly on prolongation of the treatment to 14 days, planrs treated with 50 mo l m-3 NaCI showed a cons iderable recovery of ARA with its value increas in g from 37 to 71 % of the cont ro l leve l. But this \ as not true in case of plants treated with 100 mol m-'; A RA on othe r hand furt her dec lined. Short term treatment (3 days ) of 70 day o ld pigeonpea plants al so brought about a sharp dec rease in ARAn .

It is a general observation that the processes of nodu lation and N2-fixation are more sensitive to sa­linity than growth of the host plantl.K. 192() . Decrease in

Ni -f ixat ion under salt stress has also beeB ascribed to direct effects on nitrogenase 'o.

Since saline conditi ons in the soil/rooting medium can have osmotic and nonosmotic effec ts on legume­rhizobium symbi otic system, the effect of iso-osmoti c concentrations of NaCI, mannito l and PEG on nodule

f . . h b df) 10 11 M . I I unctlOnIng ave een compare . '- . annlto so u-tions which created only osmotic stress have a much

SWARAJ & BISHNOI : SALT STRESS & NITROGEN FIXATION 845

slower effect on nodule functioning and relatively higher concentrations are required to be effective9

. It has also been reported that while salt effects are re­versible, mannitol effects lack reversibility . The in­hibitory effects of PEG on ARA are also initially much slower than corresponding salt effects but ulti­mate ly more deleterious32. In addition , the effect of these stress conditions on N2-fixation are much faster and steeper than nodule respiration . The more toxic effects of PEG as compared to NaCI are attributed to apparent penetration into the ti ssue 10 .

Preliminary studies carried out in our laboratory have shown that application of 10 11M NAA to pi­geonpea nodule slices from plants treated with 50 mM NaCI for 3 days in the suspension medium (0 .1 M phosphate buffer) containing sodium succinate, as a source of carbon skeletons during I h incubation period in the presence of C2H2 on a metabolic shaker, partially restored ARA 32. The addition of glycine be­taine ( 10 mM) to the growing medium also partially restored the activitl of young NaCI stressed alfalfa nodules probably by mainta ining high water status.

Nodule respiration Decrease in N2-fixation in sa lt stressed nodules is

accompanied by a corresponding decrease in nodule respirations.7,8.24. NaCI added directly to the incuba­tion mixture of bacteroids or to the culture medium of plants inhibits 0 2-uptake by bacteroids isolated from pea nodules . Bacteroids isolated from salt treated soybean and bean nodules show higher respiratory activity than those from pea and faba bean nodules8

in the absence of respiratory substrates . Short term application of 100 mM NaCI in nutrient

solution to the hydroponically grown soybean plants resulted in immediate inhibition of nodule respiration (Or uptake and CO2 evolution) and reduced its stimulation on raising externa l p0224. Respiratory quotient of intact nodules and fermentative metabo­lism in excised nodules increased . Such effects have been ascribed to decreased permeability of nodules to oxygen.

Oxygen diffusion resistance The regulation of oxygen supply to the bacteroids

is a very important feature of nodule functioning. The diffusion of O2 to the centra l ti ssue of the nodules is controlled by operation of a regulatory barrier33

,34.

Changes in the resistance of this barrier appear to be correlated with cell expansion and extrusion of gly-

coproteins into the intercellular spaces' s. Changes in carbohydrate supply to the- nodules often affect the degree of resistance of thi s barrier to O2 diffusion. Carbohydrates may directl / be regulating the res is­tance of this barrier either by supplying energy or osmotica5,24,26 ,

Direct evidence of decrease in nodule permeabil­itl to O2 under salt stress has been presented:l6 . How­ever, it is difficult to conclude whether decrease in nodule permeability is direct effec t of sa lt treatment or outcome of the primary effect of sa lt on some other component. The re lative increase in diffusion res is­tance by salt stress in white lupin nodules has been

37 much less than that created by other types of stress . The increase in diffusion res istance to O2 in whi te lupin nodules is accompanied by parallel increase in h I . ?7

t e g ycoprotem content- .

Proteins Salt stress leads to premature senescence of nod­

ules I9,38. Both increase in concentration of salt and duration of treatment accelerate greening of the nod­ules, which is visual indication of the ir senes-

19 18 19 Th d . I b I . cence ,. " . e ecrease m so u e protem content of nodules is a general response to sa lt stress in a number of legumes4o.

Accelerated greening of nodules under sa lt stress is accompanied by sharp decrease in the ir leghemoglo­bin (Lb) content in c hi ckpea:l~ and pea7.X which are known to be higher salt sensiti ve legumes. Cluster­bean, which is moderate ly sensiti ve to sa lt stress, showed 6-36% decrease in Lb on treatment with NaCI (50 and 100 mM) within 3-6 days after sa lt treatment. However, the decrease in Lb and tota l prote in content of nodules in response to· sa lt stress is much less in case of white lupin a re lati ve ly more to lerant symbi­otic system,

Metabolic factors Salt stress affects vari ous metabo lites (amino ac ids,

organic acids , carbohydrates) in nodule cytosol and the bacteroids23. Alfalfa plants treated for 2 weeks with 0 .15 mM NaCI have shown a 40% decrease in total organic acids except lactate which showed 11 % increase in cytosol and 94% increase in the bacter­oids2J . Malate concentration in salt stressed pea nod­ule cytosol as we ll as bacteroids is decreased7

. Salt stress induces a large increase in amino acid and car­bohydrate pools in the nodules. Among amino ac ids, proline shows the largest increase of 12 .8-fold in

846 INDIAN J EXP BIOL, SEPTEMBER 1999

nodule cytosol and 8.0-fold in the bacteroids23 and may play an osmoregulatory role. Asparagine also shows considerable increase while other amino acids show little change. Among carbohydrates, pinitol show significant increase and accounts for more than 35% of the carbohydrate pool of salt stressed nodules . This cyclitol also seems to be of osomoregulatory significance. In white lupin nodules, increasing salt stress results in increased sucrose content and de­creased starch levels27. Chickpea nodules also show some increase in total soluble carbohydrate (TSC) levels in response to NaCI application38

. Thus it ap­pears that irrespective of the salt sensiti vity of the host plant, nodules show an increase in total soluble carbohydrate level serving as an index to sa lt stress.

Among enzymes of carbohydrate metabolism in white lupin nodule, sucrose synthase shows considet­able decrease in its ac ti vity in response to salt stress27. However, acti vities of nodule cytoso lic phosphoenol­pyruvate carboxy lase and bacteroid malate dehydro­genase strongly enhanced on NaCI application in

7 pea. It has also been suggested that Nrfixation is more

sensiti ve to sal t stress than ammoni a assimilati on41. In soybean as well as Vicia faba nod ul es, the act ivities of glutamine synthetase and NA DH-GOGAT are sig­ni ficantl y reduced due to 'p resence of sa lt in the cul-

. d' 4041 Th . . . f tUring me !Urn '. e nitrogenase actiV ity was a -fec ted more sharply than acti vities of enzymes of ammoni a ass imi lation.

Activated oxygen species In addit ion to havi ng other phys iological effects,

salt stress can induce senescence process in different plant tissues by enhancing production of toxic acti­vated oxygen species (AOS) lowering the leve ls of protective enzymes li ke catalase, superox ide dismu­tase (SOD), GSSG-red uctase and ascorbate peroxi­dase. Legume nodules have a hi gh potenti al fo r pro­ducti on of AOS42.44 due to presence of proteins li ke Lb which can undergo autoox idation.

Among commonly known H20} scavenging en­zymes, catalase shows 9-47% decrease in its acti vity in nodules of chickpea'S and pigeon pea on short term treatments with 50 and 100 mM NaCI. Peroxidase activity on the other hand , show 1.5-3 .0-fold increase in nodules of all these plants. In white lup in nodules, catalase and peroxidase acti vities remain unaffected under salt stress, but SOD shows substantial decrease in its activity .

Enzymes of ascorbate-glutathi one cycle, which serve as alternative H20 2 scavenging system in nod­ules45 also show some decrease in their activity under saline conditions. Short term treatment of plants with NaCI (50, 100mM) results in 13- 15% decrease in ASC-peroxidase of chickpea nodul esJX and 6-29% decrease in c1usterbean nodules. However, after 7 days of salt treatment, the acti vity of ASC-peroxidase in chickpea nodules starts recovering reaching the control value by 14 days. Adverse effect of salt stress on GSSG-reductase appears latter than th at on ASC­peroxidase. Some decrease in GSSG-reductase ac ti v­ity in chickpea nodules could be seen onl y after 7 days of NaCI treatment's.

NaCI induced senescence of nodu les and decrease in Nr fi xati on is assoc iated with simultaneous de­crease in the levels of anti oxidanL like ascorbi c ac id (ASC) thus shi fting the redox level of nodules to­wards the more ox idative side. Hi gher leve l of st ress ( 100 mM NaCI) brings down ASC con tent of cluster­bean nodules by 23-34% with in ~-6 days of the treat­ment28.

Decrease in the acti vity of H}O} scavenging en­zy mes and level of antiox idants in the sa lt stressed pigeonpea nodul es refl ects itself in H20 1 accumula­tion' 2. On Icachi ng the sa lt out of the root ing medium, H}02 content in the nodul es grad ua lly starts dec lin­ing. Increased perox ide leve ls of the sa lt stressed nodu les is also associated with increased leve l of li pid perox idation12 which poi nts towards increased membrane disorgani sati on. On removal of the salt from the rooting medium, li pid perox idarion also starts graduall y dropping.

Nodule structure High salt concentrat ions in the rooti ng med iulll

brings about a nu mber of changes in the ultra- struc­ture of nodules. Alterati ons in nodul s after 2h expo­sure of soybean plants to 100 mM NaCI inc lude a lobed nucleus with di ffe rent chromat in distri but ion , enl arged peripl as l11ic space and appearance of wall in fo ldingJ6. Structural changes also occur in endo­plasmic reticulum, microtubules . mitochondria and plastids. Light microscopy of these nod ules show de­crease in the area of inner cortica l cells1(, .

The intensity of deleterious effec t of sa lt also de­pends on growing conditions of plants. ft has been seen that soybean nodul es receiving adequate supply of photosynthates under hi gh irradiance could tolerate and recover from long term effects of sa lt with litt le

SWARAJ & BISHNOI: SALT STRESS & NITROGEN FIXATION 847

alterations in their structural integrit/6. However,

Zaharan and Sprent46 working with indeterminate faba-bean nodules have observed that adverse effects of salt on N2-fixation are much higher when plants are growing actively during summer under high irra­diance than when they are growing slowly during winter under low irradiance46

. Some of the soybean nodules harvested from plants grown under low light intensity 3 weeks after salt treatment showed onset of meristematic activity in cortex; young cells here con­tained infection threads and newly released bacteria. The reversal of determinant state in soybean nodule from plants grown under low light intensity 2 weeks after NaCI application is thought as an attempt to pre­serve some level of nodule activity when rapid recov­ery is not possible. Enhanced meristematic growth of faba-bean indeterminate nodules on subjecting the plants to 100 mol m·3 is also looked upon as a com­pensatory mechanism to cope with low nodule acti-

. 19 Vlty .

It has been suggested that cortex may act as an !on barrier in soybean, faba-bean and common .bean nod­ules as much less chloride and sodium ions are ob­served in the infected tissue of the nodule as com­pared to its cortex47

. It is possible that process of ex­clusion of Na+ and cr operates in the inner cortex of most legume nodules37 and in salt sensitive symbiotic systems, this phenomenon can switch on functioning of osmocentraclile cells thus increasing the resistance to O2 diffusion24

.

X-ray microanalysis of white lupine nodules have shown that following 6d exposure to 150 mol m-3

NaCl, sodium ions are largely excluded from the in­fected zone of the nodule and only low levels of chlo­ride ions penetrated into this region27

• This is consid­ered as one of the reasons for relatively higher toler­ance of this symbiotic system to salt stress.

Future prospectives Concerted efforts must be made to optimize contri­

bution of symbiotic Nr fixation towards plant growth and yield especially in soils infested with high salin­ity where it is not possible to apply any nitrogenous fetilizers. This can be achieved by developing symbi­otic systems in legumes which can withstand exces­sive salt concentrations in the soil solution by devel­oping one or more of the following characters: (i) Nodules should be capable of bringing about

appropriate compartmentation of the absorbed ions in the cortical region so that toxic ions are

unable enter to Nrfixing control tissue. (ii) Maintain or reinstate the nodular meristematic

activity so that the damaged symbiotic ti ssue can be replaced with the new one for longer du­ration at the time.

(iii) Nodules should be able to make osmotic ad­justment by accumulating metabolically com­patible solutes.

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