654 SHORT COMMUNICATIONS VOL. 2 6 (1957)

On the relation between nitrogen fixation and nodule nitrate reductase of soybean root nodules*

I t h a s been repor ted I t h a t Rhizobium cells f rom nodules of cer ta in l egumes con ta in a n i t r a t e r educ tase . There ha s been no evidence, however , to indicate w h e t h e r or no t th i s e n z y m e is physiological ly i m p o r t a n t in t he n i t rogen-f ix ing process. I t h a s been suggestedZ, 2 t h a t n i t r a t e or ni t r i te , poss ib ly fo rmed f rom t h e decompos i t ion of t he init ial p roduc t s of f ixation, m a y induce t h e adap t i ve fo rma t ion of t he enzyme. There is also t he poss ibi l i ty t h a t t he nodule n i t r a t e r educ t a se is non-specific for e lec t ron acceptor a n d t h a t n i t r a t e r educ tase is real ly a misnomer . This possibi l i ty t oge t he r wi th o ther detai led proper t ies of t h e s y s t e m are being inves t iga ted a t t h e p re sen t t ime and will be repor ted in detai l a t a la ter date.

I t is well k n o w n t h a t different s t ra ins of Rhizobium of a g iven inocula t ion group m a y v a r y in the i r effect iveness to fix molecular n i t rogen dur ing symbios is . VIRTANEN et al.a, 4 have shown a close correla t ion be tween t he ni t rogen-f ix ing capac i ty of p l an t s and t h e hemoglob in concen t ra t ion in t he roo t nodules . Recent ly , HAMILTON, SHUG AND WILSON 5 h a v e provided evidence regard ing t h e mode of ac t ion of hemoglob in in n i t rogen f ixat ion.

The e x p e r i m e n t s repor ted in t h e p re sen t c o m m u n i c a t i o n d e m o n s t r a t e t h a t the n i t rogen-f ix ing capac i ty of t he soybe an p lant , t h e n i t r a t e r educ tase ac t iv i ty of nodules , a n d t he hemoglob in con ten t of nodules are all posi t ively correlated.

I n these expe r imen t s four selected s t r a ins of Rhizobium japonicum, kind ly provided by Dr. L. W. ERDMAN of t he U.S. D e p a r t m e n t of Agr icul ture , were cu l tu red on s l an t s con ta in ing a mann i to l - yea s t ex t r ac t m e d i u m . Soybean seed (Glycine max. Merr. var. Lee) were dis infected by a five m i n u t e immer s ion in "Ch lo rox" (5% s o d i u m hypochlor i te) and t h e n were washed by decan ta t i on wi th steri le disti l led wa t e r to r emove t he d is infec tan t . T he washed-d is infec ted seeds t h e n were inocula ted wi th a wa t e r w a s h f rom s l an t s t h a t h a d been cu l tu red for t en days .

The m e t h o d for t e s t i ng cu l tures of Rhizobium for n i t rogen-f ix ing efficiency was an a d a p t a t i o n of t h a t descr ibed by LEONARD e. The modif ica t ions cons is ted of t h e following : (i) K N O 3 was omi t t ed ent i re ly f r om t h e n u t r i e n t solut ion; (2) Na2MoO 4 was included in t he n u t r i e n t solut ion a t a concen- t r a t i o n of o.o2 p .p .m. ; (3) one-hal f inch of steri l ized coarse gravel was applied to t he surface of t he s and cu l tu re to reduce evapora t ion and c o n t a m i n a t i o n * * and (4) a layer of sterile co t ton was placed on t he sur face of t h e gravel af ter emergence of t he seedlings to fu r the r reduce possible c o n t a m i n a - t ion. T h e p r epa ra t i on of t h e cu l tu res was conduc ted in a dis infected inocula t ing chamber .

The cu l tu re assembl ies , each con ta in ing four p lan ts , were repl icated four t imes and r andomized on a bench in t h e greenhouse . P l a n t s were h a r v e s t e d af ter an e igh t -week cul ture period. The tops of p l an t s were cu t a t t h e level of t he s and and t he roots were careful ly r emoved and all nodules collected as descr ibed by EVANS 1. All s u b s e q u e n t work was carr ied ou t a t o to 4 °. The h a r v e s t e d nodu les were washed in cold disti l led water , b lo t ted d ry and homogen ized (by use of a m o r t a r a n d pestle) in 4 weights , per weight of nodules , of o.oo 5 M p h o s p h a t e buffer a t p H 7-5. The h o m o g e n a t e was squeezed t h r o u g h 4 layers of cheesecloth a n d t h e n centrifuged, a t 3o,ooo × g for 5 rain. The s e d i m e n t con ta in ing t he bacter ia l cells was r e suspended wi th t he a id of a Ten Broeck homogen ize r in t h e original vo lume of o .oo5M p h o s p h a t e buffer a t p H 7-5- Th i s suspens ion was used for t he n i t r a t e r educ tase assays .

The comple te a s s ay m i x t u r e for n i t r a t e r educ tase cons is ted of the following: i o / zmoles p h o s p h a t e buffer p H 7.5; IO/zmoles NaNO3; io /*moles succ ina te and o.I or o.2 ml of t he bacter ia l suspens ion con ta in ing 1.2 to 6.0 m g pro te in /ml . T he final vo lume was ad ju s t ed to 0.5 ml wi th water . The p rocedure for t he n i t r a t e r educ tase a s s ay was t h a t descr ibed by EVANS 1 wi th t h e except ion t h a t succ ina te was used as an e lect ron donor ins t ead of reduced d iphosphopyr id inenuc leo t ide .

The s u p e r n a t a n t f rom t he cen t r i fuga t ion was used for hemoglob in de t e rmina t i on which was e s t ima t ed as t h e pyr id ine h e m o c h r o m o g e n as described by KEILIN AND HARTREEL The s t a n d a r d pyr id ine h e m o c h r o m o g e n was p repared f rom recrystal l ized h e m i n (Nutr i t ional Biochemicals Company) . I n ca lcula t ing t he hemoglob in con t en t of nodules t he absorp t ion of t he pyr id ine solut ion f rom the N-I (Table I) nodules which con ta ined no hemoglob in was sub t r ac t ed f rom the absorp t ion of t he pyr id ine solut ion f rom nodules of p l an t s inocula ted wi th t he o ther s t ra ins .

T h e tops of t he p l an t s were dried in a forced d ra f t oven a t 80 ° for 24 h, t h e n g round in a Wi ley mill. To ta l n i t rogen con t en t was de t e rmi ned by t he Kje ldah l me thod . The pro te in con t en t of t h e bacter ia l suspens ions was e s t i ma t ed by to ta l N × 6.25.

The m e a n va lues for t he d ry weigh ts of soybean tops, to ta l N con t en t s of tops, f resh weigh ts of nodules , hemoglob in con t en t s of nodules and n i t r a t e r educ tase ac t iv i t ies of nodules are p resen ted in Table I. As indica ted by t he da ta , t h e yields and to ta l N con ten t s of tops var ied widely ind ica t ing

* Cont r ibu t ion of t he Divis ion of Biological Sciences, Nor th Carol ina Agr icul tura l E x p e r i m e n t Stat ion. Th i s work was suppor t ed in pa r t b y a g r a n t f rom t h e Na t iona l Science F o u n d a t i o n (No. G 2,653).

** Sugges ted by Dr. L. W. ERDMAN in a p r iva t e commun ica t i on .

VOL. 2 6 (1957) SHORT COMMUNICATIONS 6 5 5

T A B L E I

T H E E F F E C T OF Rhi*obium STRAIN E F F I C I E N C Y ON DRY W E I G H T AND TOTAL N I T R O G E N OF

SOYBEAN TOPS AND ON T H E HEMOGLOBIN C O N C E N T R A T I O N , N I T R A T E R E D U C T A S E ACTIVITY

AND F R E S H W E I G H T OF N O D U L E S

Enttyrae activ~y Dry weight ot Total N Fresh weight o/ Hemoglobin (l~moles NO=/xo min/mg protein) nodules

Strain tops (g/culture) o/tops (%) (g/culture) (l~g/g /resh nodule) Without succinate With succinate

N" I 1 . 5 8 0.93 0.85 o.o o.o o.o N 2 2.57 2.53 1.64 72.4 ° 23.66 77.83 N 3 5.15 3.72 1.53 145.52 81.oo 12o.o 7 N 4 3.77 3-48 1.23 122.oo 62.85 1 lO.68

L.S.D. (0.05) 0.92 o.21 N.S. 17'73 44.34 33-74 L.S.D. (o.oi) 1.39 0.30 N.S. 26.86 N.S. N.S.

The correla t ion coefficients, ind ica ted wi th a single as te r i sk where s ignif icant a t t h e o.o 5 level a n d wi th a double as te r i sk where s ignif icant a t t he o.oi level, were as follows: to ta l N vs. hemo- globin conten t , o.96**; to ta l N vs. e n z y m e ac t iv i ty wi th succinate , 0.68*; to ta l N vs. e n z y m e ac t iv i ty w i t h o u t succinate , o .68"; hemoglob in con t en t vs. n i t r a t e r educ tase ac t iv i ty wi th succinate , o.71 **; hemoglob in con t en t vs. e n z y m e ac t iv i ty w i t hou t succinate , o.69"; d ry weight of tops vs. e n z y m e ac t iv i ty wi th succinate , o.68 * ; d ry weight of tops vs. e n z y m e ac t iv i ty w i thou t succinate , o. 54-

The d a t a ind ica ted for t he var ious m e a s u r e m e n t s are m e a n s for t he four repl icates of t he exper imen t . The least s ignif icant differences (L.S.D.) for t he compar i son of a n y two m e a n s are indicated. An L.S.D. was no t ca lcula ted unless t he F va lue showed an overal l s ignif icant effect of s t ra in . E v e n thougrl t he overal l effect of s t r a ins was significant , in cer ta in cases no t all of t he pos- sible ind iv idua l compar i sons were s ignif icant .

different n i t rogen-f ix ing efficiencies of t he va r ious Rhizobium s t ra ins . The overal l t r e a t m e n t effect of s t r a ins on d ry weigh ts of p l a n t tops, to ta l N con t en t s of tops and hemoglob in con ten t s of nodules was s ta t i s t i ca l ly s ignif icant a t t h e o.oi level as ind ica ted b y F values. The overall t r e a t m e n t effect of s t r a ins on n i t r a t e r educ tase ac t iv i t ies were s ignif icant a t t he 0.05 level. As indica ted in Table I, to ta l N c o n t e n t s of tops were pos i t ive ly corre la ted wi th hemoglob in con t en t s and e n z y m e act iv i t ies of nodules . Bo th hemoglob in c o n t e n t s of nodules and d ry weigh ts of tops were pos i t ive ly corre la ted wi th nodule n i t r a t e r educ tase act ivi t ies .

F r o m the above resul ts , i t seems clear t h a t nodule n i t r a t e r educ tase ac t iv i ty is pos i t ive ly corre la ted wi th n i t rogen f ixat ion capac i ty . F u r t h e r e x p e r i m e n t a t i o n is in progress which is de- s igned to de te rmine w h e t h e r or no t t he re la t ionship is direct .

G E O R G E M . C H E N I A E North Carolina State College, School o/Agriculture, Raleigh, N.C. (U.S.A.) HAROLD J. EVANS

I H. J. EVANS, Plant Physiol., 29 (1954) 298. G. M. CHENIAE AND H. J . EVANS, in W. D. MCELROY AND B. GLASS, Inorganic Nitrogen Metabo- lism: Function o/Metallo-Flavoproteins, J o h n s Hopk ins Press , Bal t imore , 1955, p. 184.

3 A. I. VIRTANEN, J. JORMA, H. LINEOLA AND A. LINNASALMI, Acta Chem. Scan&, I (1947) 9o. 4 A. I. VIRTANEN, J. ERKAMA AND H. LI~rEOLA, Acta. Chem. Scan&, I (1947) 861.

P. B. HAMILTON, A. L. SHUG AND P. W. WILSON, Proc. Natl. Acad. Sci. U.S., 43 (1957) 297. s L. T. LEONARD, J . Bacteriol., 45 (1943) 523.

D. KEILLIN AND E. F. HARTREE, Biochem. J. , 49 (1951) 88. Rece ived A u g u s t 24th, 1957