Plan de manejo ambiental

In ro o t n o d u le s, th e firs t p ro d u c t o f n it r o g e n fix a tio n a m m o n ia is e x c re te d f r o m th e b a c te ro id s a n d th e n a s s im i la t e d b y th e p la n t. O n ce fo rm e d , a m m o n ia c o u ld b e a s s im ila te d b y v a r i o u s p ath w ay s. F o r m an y y ea rs the as s im ila tio n o f am m o n ia w as th o u g h t to b e only v ia th e red u ctiv e am in atio n o f 2 - o x o g lu ta ra te to g iv e g lu ta m a te a s ca ta ly z e d b y g lu tam a te d eh y d ro g en a se (G D H , E C 1.4.1.3.).

G D H

2 - o x o g lu ta ra te + N H 3 +NAD(P)H +H+ --- » glu tam ate +

NAD(P)+ + H20

G D H a c tiv ity h as b e e n fo u n d in a n u m b e r o f p la n t sp ecies a n d is a b u n d a n t in m o s t tiss u e s . In itia l s tu d ie s o f K e n n e d y (1 9 6 6 ) on S e rrad ella n o d u le s su g g ested th a t th e a m m o n ia f r o m n itro g en assim ilatio n w as in c o rp o ra te d d ire ctly in to g lu ta m a te . H o w e v e r its h ig h K m f o r N H 3 a n d its v irtu a l d isap p e aran ce in K le b sie lla a e r o g c n e s u n d er c o n d itio n s o f lo w N a v a ila b ility w h en N H 3 a s s im ila tio n w a s s till o ccu rrin g , re s u lte d in th e d is c o v e ry o f a n a lte rn a tiv e ro u te o f a m m o n ia a s s im ila tio n in th is o rg an ism in 1 9 7 0 , co m b in in g g lu ta m in e s y n th e ta s e (G S ) a n d a n ew ly d isco v e red e n z y m e g lu tam ate s y n th a s e (G O G A T E C 2 .6 .1 .5 3 .X T e m p e s t e t

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a l. 1 9 7 0 ). M iflin a n d L c a (1 9 7 6 ) s u g g ested th a t a s s im ila tio n o f am m o n iu m o c c u r s in the p la n t c y to so l b y th e G S /G O G A T p a th w a y , ra th e r th a n b y G D H , fo llo w in g a re a n a ly sis o f K en n e d y 's d a ta fro m lab ellin g o f S e r r a d e lla n o d u le s. M o re v e r M iflin an d L e a s u p p o se d th a t G S /G O G A T p a th w a y is the m a jo r, perh ap s o n ly ro u te o f a m m o n ia a s s im ila tio n in h ig h er p la n ts (1976). In th is p ath w ay G S is resp o n sib le f o r th e in itia l assim ilation o f a m m o n ia in to g lu ta m in e a n d G O G A T c a ta ly z e s th e tra n sa m id a tio n o f g lu ta m in e a m id o -n itro g e n to th e a - a m i n o p o s itio n o f 2 - o x o g lu ta ra te to f o r m tw o m oles o f g lu tam a te. B ec au se th e tw o e n z y m e s a r e dependent o n e a c h o th e r fo r th e p ro v is io n o f su b strate, th e ir a c tiv itie s c o n s titu te a cy cle w h ic h has been te rm e d th e g lu tam ate sy n th ase c y c le (M iflin a n d Lea 1980).

(G S)

G lu ta m a te + A T P + N H 3 --- ► G lu ta m in e + A D P P i + H 2O

2 -o x o g lu ta r a te + G lu ta m in e + N A D H ( o r N A D P H o r F d red. )+

(G O G A T )

---► 2 G lu ta m a te + N A D + ( N A D P + o r F d o x . )

M ore e le g a n t studies b y M eek s e t a /.(1 9 7 8 ) o n s o y b e a n n o d u le s u s in g 13N 2t.a s h o r t- liv e d rad io activ e iso to p e of N, d e m o n stra te d that N H 4 + is f ir s t in c o rp o rated in to th e am id e p o sitio n o f g lu ta m in e i n the re a c tio n c a ta ly z e d b y G S . T h is in co rp o ratio n is b lo c k ed b y m e th io n in e sulfoxim ine. a n in h ib ito r o f G S (M eek s e t a l., 1978). T h e a m id e g ro u p is su b seq u en tly tr a n s f e r r e d to th e 2 - c a r b o n o f o x o g lu ta ra te in th e r e d u c tiv e am in atio n

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re a c tio n c a r r i e d o u t by G O G A T , w h ich is b lo c k ed b y a z a se rin e , a general in h ib ito r o f a m id e - g r o u p tr a n s f e r r e a c tio n (M eek s c t al. 1 9 7 8 ). S im ila r re su lts h a v e b e e n obtained b y O h y am a a n d K u m az aw a (1 9 8 0 ). B e c a u s e GS h a s a m u c h lo w e r Km fo r a m m o n ia th a n G D H , th e g lu tam a te s y n th a s e cycle c a n fu n c tio n a n d m aintain v e ry low c o n c en tratio n s o f a m m o n ia in th e tissue a n d w o u ld b e im p o rtan t to m a in ta in a g ra d ie n t fav o u rin g c o n tin u e d N H 4+ e x p o rt b y b a c te r o id s ( R a w s th o m e c t al., 19 8 0 ). F u rth e r e v id e n c e f o r the G S / G O G A T p ath w ay as th e p rim ary a m m o n ia as s im ila to ry r o u te com es f ro m th e d e v e lo p m e n ta l s tu d ie s o f n o d u le en z y m e a c tiv itie s . R o b e rtso n c t al. ( 1 9 7 5 a ,b .) rep o rted th a t b o th G S an d a n N A D H -d e p e n d e n t G O G A T are in d u c e d d u r i n g lu p in n o d u le d e v e lo p m e n t in p a r a lle l w ith n itro g e n a s e a c tiv ity a n d le g h e m o g lo b in c o n te n t a n d in a lfa lfa b o th G S a n d G O G A T d e c lin e a lo n g w ith n itro g e n a s e activ ity w h e n n o d u la tin g ro o ts a re d ep riv ed o f th e ir s h o o t sy stem . T h e s e e n z y m e s in c r e a s e in a c tiv ity a g a in as n itro g e n a s e a c tiv ity in c re a s e s d u rin g n o d u le re b u ild in g (G ro a t a n d V ance, 1 9 8 1 ), y e t c h a n g e s in th e a c tiv ity o f G D H d o n o t a p p e a r to b e c o rrelate d w ith n i t r o g e n fix a tio n . D u r in g n o d u le d e v e lo p m e n t in s o y b e a n s G D H a c tiv ity d e c lin e d m ark ed ly a t a tim e w h e n n itro g en fix a tio n w a s in creasin g (S e n an d S c h u lm a n 1980).

In v ie w o f all th e a b o v e e v id e n c e , it is co n v in c in g th a t in legum e r o o t n o d u le s am m o n ia e x c re te d by th e b a c te ro id s is assim ilated o n ly v ia the g lu ta m a te sy n th a s e cycle.

W o r k o n o th e r p a r ts o f th e p la n t h a s a lre a d y s h o w n th a t the g lu ta m a te s y n th a s e cy c le is th e m a jo r ro u te o f a m m o n ia a s s im ila tio n in h ig h e r p la n ts . A m m o n ia in h ig h e r p la n ts is d eriv ed b o th f r o m th e p la n t's

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p rim a ry n itro g e n s o u r c e s (am m o n ia, n itrate o r , f o r le g u m es, d in itro g en ) a n d f ro m a n u m b e r o f in te rn a l n it r o g e n - c y c l in g p a th w a y s s u c h as p h o to resp iratio n , a m in o acid catab o lism and p h e n y l p ro p an o id m e tab o lism . T h e g lu tam a te s y n th a s e cycle is th e re fo re e s s e n tia l n o t o n ly f o r p rim a ry n itro g e n a s s im ila tio n b u t also to m a in ta in th e g e n e ra l n itro g e n ec o n o m y o f th e p lant.