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I t l I I _ _ ®

Fig. 1. Schcmatic representation o f

thc th ree processes induced by

Rhizobium in legume roots. (1) root

hair deformation and curling, (2)

infection thread formation, and (3)

mitotic activity in the root cortex.

the s igna ls tha t t r igg er the form at ion o f the nodule orga n .

Relatively few nodulin genes have been identif ied that are ex-

pressed in the deve loping root nodule wel l before the onse t of

n i t rogen f ixa t ion . The pro te ins f rom these ear ly nodul in genes a re

mo st l ike ly i0volved in the infec t ion process and deve lopm ent o f the

nodule s t ruc ture . Most ear ly nodul ins a re h ighly pro l ine- r ich pro-

te ins , and m ay therefore be ce ll wal l comp onents (8). The induct ion

of p lan t ce l l d i f fe ren tia t ion and of ear ly nodul in gene express ion i s

mo st l ikely und er con trol o f the bacterial pa rtner.

I n n u m e r o u s R h i z o b i u m species, genes involved in establishing an

effect ive symbios i s have been ident if ied wi th the use o f t ransposon

mutagenes is , complementa t ion ana lys i s , and o ther gene t ic tech-

niques (7, 11, 12) . In the fas t -growing R h i z o b i u m species, most of

these genes a re loca ted on la rge p lasmids (pSyms) , whereas the

s l o w - g r o w i n g B r a d y r h i z o b i u m species carry these genes on the bac-

ter ia l chro moso me. G enes essentia l for the process of n i t rogen

f ixa tion inc lude n~and f ix , am ong w hich a re the s t ruc tura l genes for

n i t rogenase (13) . Desp i te the in t r ins ic impor tance o f these genes for

nitrog en fixation, n/f and f i x mutants a re ab le to induce the

format ion o f root nodu les in w hich a ll nodul in genes a re expressed

(8) and wi th a mor pho logy l ike tha t of nodules induced by wi ld- type

R h i z o b i u m . Apparen t ly , n i t rogen f ixa tion i s the resu l t o f and no t a

prerequis i te for nodule deve lo pme nt and nodul in gen e express ion .

The rh izobia l genes requi red for nodule format ion and nodul in

gene express ion inc lude the nod ula t ion n o d ) genes, several groups

of genes concerned wi th the s t ruc ture o f the outer sur face of the

bac te r ium ( the exo, lps, a n d ndv genes) , and a num ber of l ess wel l

defined genes 8, 12) . Fo r m a l p r o o f f o r th e i n v o l v e m e n t i n t h e

induct ion o f the express ion o f early nodul in genes has only been

demonst ra ted for the n o d genes (8) . Up on t ransfer of the nod gene

region , the rec ip ien t non-nodula t ing Agrobacter ium gained the abil i ty

to nodula te . The nod gene products , therefore , a re the most l ike ly

candida tes to be s igna ls e l ic i ting no dule de ve lopment .

T h e nod genes fal l in three classes, com mo n, host-specific, and

n 0d D. T h e c o m m o n nod genes inc lude , among o thers , the n0dABC

genes tha t a re found in a ll rh izobia l species . The c om mo n nod genes

of different species are struc turally very similar a nd functiona lly

in te rchangeable (7 , 12) . Muta t ions in the n o d A B C genes abol i sh

comple te ly the ab i li ty to nodula te , which under l ines the i r p ivota l

ro le in nodule deve lopment .

The host-specific nod genes de te rm ine the speci fici ty o f nodula t ion

o n a p a rt ic u la r h o s t. U p o n m u t a t i o n o f s u c h a nod g e n e , n o d u l a t i o n

is delayed or reduced , or the h ost ran ge is al tered. H ost-spec ific nod

genes a re not func t iona l ly in te rchangeable be tween rh izobia l spe-

c ies , desp i te a somet imes s t r ik ing sequence conserva t ion be tween

the respective genes I 1 ) . A l t h o u g h s e q u e n c e h o m o l o g i e s a n d

prote in ana lyses o f No d pro te ins h in t a t a d ivers i ty of func t ions ,

conclusive evidence for any biochemical function is lacking (12).

The th i rd type o f n od gen es is n0dD (7, 12) , of which som e species

car ry mul t ip le copies . The n0dD gene i s the only nod gene tha t i s

consti tut ively expressed in bot h th e free-l iving and sy mb iotic states

o f R h i z o b i u m . In combina t ion wi th f lavonoids excre ted by p lan t

roots , the NodD pro te in probably ac t s as a t ranscr ip t iona l ac t iva tor

for al l other nod genes, and the gene is as essential for nodulation as

a r e t h e c o m m o n n 0d A BC g e n es . Be c a u se th e N o d D p r o t e i n o f a

1 6 N O V E M B E R 1 9 90

particular R h i z o b i u m species proves most responsive to the f la

vonoids excre ted by i t s homologous hos t , NodD a lso cont r ibutes t

host specificity.

R o o t

Hair Deformation and Curling

The f ir st m icroscopica lly vi sib le responses of the legume u po

contac t wi th R h i z o b i u m are deformat ion and cur l ing of root ha i r

14) . Rhizobia in te rac t wi th a lmost a l l the young emerging roo

hair s , bu t on ly -2 5 % of the root ha ir s ac tua l ly curl . Com pute

simulation studies suggest that the curl ing is caused by loca

s t imula t ion of grow th o f the roo t ha i r tip , ra ther than by inhib i t io

o f g r o w t h I4 ) . Ev en at this early stage, no dula tion is host-specific

because as a ru le only the homologous R h i z o b i u m causes deforma

tion and curl ing. Because R h i z o b i u m i s chemotac t ic towards th

f lavonoid ab le to induce nod gene expression (15), host specificit

may even be m ani fes ted in the rh izosphere before p lan t an

bacterium visibly interact .

Plan t gene express ion cor re la ted wi th deformat ion and cur l ing i

d i ff icu lt to approach exper imenta l ly . On ly in one s tudy have change

in the express ion level of two p lan t genes been dem onst ra ted ( I6)

and n o p lan t genes have ye t been ana lyzed in fur ther de tai l.

Wi th respec t to R h i z o b i u m , i t was shown over 20 years ago tha

the physical p resence of the bacteriu m is not a prerequisi te for roo

hai r deformat io n ( •7) . M ore recent ly , i t was shown tha t s te r il

cu l ture f il trates o f rh izobia grow n in the presence of root exudate o

nod gene- inducing f lavonoids conta in fac tors tha t cause root ha

defbrmat ion . Genet ic ana lyses ind ica ted tha t the presence of f im

t i o n a l c o m m o n nod genes i s required fo r the product io n of thes

extracellular facto r (7). R ecently , D~nari~ and co-w orkers hav

purif ied an extracellular co m po un d excreted by the alfalfa sym bion

R . m e l i l o t i and elucidated i ts structure 1 8 ) . T h i s c o m p o u n d

NodRm-1, i s shown by chemica l ana lys i s to be a su lpha ted [3-1 ,4

te t rasacchar ide of D-glucosamine , in which three amino groups a r

ace ty la ted and one i s acyla ted wi th an unsa tura ted Cl6 fa t ty ac i

cha in (Fig . 2 ) . Because the func t iona l ly in te rchangeable commo

nod genes a re involved in the synthes is of N od Rm -1, i t is l ike ly tha

deformat ion fac tors produced by o ther rh izobia l spec ies wi l l hav

f e a t u r e s i n c o m m o n w i t h No d Rm - 1 .

N o d R m - ] is o n ly e xc re te d w h e n t h e c o m m o n nod genes and

host-specific nod gene (n0dH) a re func t iona l and the R. m e l i lo

bac teria a re grown in the presence of the nod g e n e - i n d u c i n

f lavonoid lu teo l in . Ad di t ion o f pur i f ied N od R m -] to a lfa lfa roots i

a concent ra t ion range o f 10 -8 to 10 - l~ M resu lted in root ha

deformat ion , whereas no deformat ion was observed when th

c o m p o u n d w a s a d d e d t o v e t c h r o o t s 1 8 , 1 9 ) . A R . m e l i lo ti n o d

mu tant i s unable to cause roo t ha i r deformat ion on a l fal fa , bu t doe

induce roo t ha i r deforma t ion o n ve tch . Analysi s of the nodH m u t a n

s h o w s i t t o b e d e f e c t i v e i n No d Rm - I p r o d u c t i o n . I n s t e a d , t h

mutant excre tes a more hydrophobic fac tor , NodRm-2, tha t , i

/OS03H

CH20H CH20H CH;OH CH 2

ao'~ N,H, NO'' N,H' NO'' N,H' HO'' N,H

CO CO CO CO

C-H i I I

CH 3 CH 3 CH 3

/ /

H-C,

CH

II

CH

Fig. 2.

Structure of the N odR m- I factor that is excret

(~H2ls by thc alfalfa sym bion t R, m d i lo ti upon induction of t

CH3

nod gcncs 1 8 ) .

A R T I C L E S 9

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pur i f ied form, causes root ha i r deformat ion on ve tch but no t on

a lfa lfa . The s t ruc ture o f N od R m -2 i s identica l to N od R m- 1, except

f o r th e p r e s e nc e o f th e s u l p h a t e g r o u p . T h e N o d H p r o t e i n m a y t h u s

be a specific sulphate transferase (19).

T h e d i f f e r e n c e i n s t r u c t u r e b e t w e e n No d Rr n - 1 a n d No d Rm - 2

shows tha t min or s t ruc tura l fea tures in the o l igosacchar ide molecule

can be respons ib le for the a l l -or -noth ing p henotype . I t a l so sugges ts

that at least some host-specific nod genes a re involved in modi fy ing

a bas ic com pou nd to form a s igna l molecule tha t i s spec if ic for the

hom olog ous hos t . The avai lab il ity of pur if ied NodR m-l ik e fac tors

and m utants for a l l nod gene s is l ikely to result in a rapid e lucidation

of the b iosynthe t ic pa thwa y for these fac tors and the func t ions o f the

var ious nod genes . I t wi l l be in te res t ing to see whether NodRm-l ike

fac tors are formed by deg rada t ion of ex is ting ce l l wall m acrom ole-

cules , o r by synthes i s f rom glucosamine res idues . Moreover , the

availabil i ty of purif ied N od factors wil l facili tate research in to the

s igna l t ransduct ion pa thways tha t l ead to nodule deve lopment and

nodu l in gene express ion .

Infection

Upon root ha i r cur l ing , rh izobia become ent rapped in the cur l s .

The resu l ting h igh loca l concent ra t ion of bac te ria l fac tors may be the

decisive step for the in duc tion of infection, because inf ection is also

observed in roo t ha i rs tha t e n t rap bac te ria whi le they tou ch ins tead

of curl (14). Actual infection starts w ith a very localized h ydrolys is of

he p lan t ce l l wall in the roo t h a i r cur l. T he mech anism of th i s

hydrolys i s i s no t c lear. The bac te r ia may produce hydroly t ic enzym es

that are responsible for the cell wall dissolution. Alternatively, the

bac te r ia may enl i s t endogenous p lan t mechanisms, such as , for

ins tance , those used wh en epidermal ce l l s form r oot ha i r s 14).

At th e si te o f cell wall hydro lysis, bacteria enter the ro ot h air cell

by invagina t ion of the p lasma mem brane . Aro und the invag ina ted

membrane , the p lan t forms a tube- l ike infec t ion thread by depos i -

t ion of ce ll wall- l ike material 4, 7) . As the infec t ion thread

penetrates the root hair cell , the bacteria proliferate within the

thread and become sur rounded by a mucopolysacchar ide mat r ix .

Most l ike ly both bac te r ia l and p lan t components cont r ibute to th i s

mat r ix, a l thoug h as ye t a p lan t -der ived or ig in has been demo nst ra ted

only for one g lycoprote in (20) . The infec t ion threads car ry the

bac te r ia f rom the root ha i r to the nodule pr imordia formed in the

root cor tex .

Deta i led cy to logica l ana lyses of the pea r oot cor tex in to wh ich

infec t ion threads gro w and ramify show tha t d i st inc t morpholo gica l

changes occur in cor tex ce l l s before they a re pene t ra ted by an

infec t ion thread 14). Microtubules rear range , the nuc leus migra tes

to the ce l l cen ter , and an add i t iona l ce l l wall i s formed. T hese

morphologica l changes sugges t tha t a s igna l f rom the growing

infec t ion thread prepares the cor t ica l ce l l s for pene t ra t ion . As the

morphologica l changes resemble changes observed in the in i t i a l

steps of the cell cycle, i t has bee n sug gested that th e co rtex cells are

start ing cell division an d are arrested in the prop hase of the cell cycle.

The expression of two ear ly nodul in genes has been cor re la ted

wi th the infec t ion process in pea . The ear ly nodul in cDNA c lones

p P s E N O D 5

21)

a nd p P s E N O D 1 2

22)

were i so la ted f rom a pea

nod ule cD N A library by differential screening. Sequ ence analysis

shows both ear ly nodul ins to be pro l ine- r ich pro te ins . The

Ps E NO D1 2 e a rl y n o d u l i n i s fo r th e m a j o r p a r t c o m p o s e d o f t w o

repea t ing pentapcpt ides , each of which conta ins two pro l ines. This

s t ruc ture sugges ts tha t th i s ear ly nodul in i s a hydroxyprol ine- r ich

cell wall prote in 22). T h e Ps ENOD5 p r o t e i n , b e s i d e s b e i n g r i c h i n

proline, is relatively r ich in alanine, glycin e, and serine, sug gestin g i t

i s re la ted to a rab inogalac tan pro te ins 21). P s E N O D 5 l a c k s t h e

r e p et it iv e s t r u c t u re o f Ps E NO D1 2 . Bo t h e a rl y n o d u li n s h a v e

puta t ive s igna l pept ide a t the am ino te rmina l end o f the i r sequenc

and therefore a re probably t ranspor ted across a membrane . In s i t

hybr id iza t ion s tudies wi th the use of an t isense R N A probes sho

tha t the P sE N O D 5 gene i s on ly expressed in ce ll s conta in in

growing infec t ion threads 21) (Fig. 3, C and D). Becaus

arabinogalac tan- like pro te ins a re know n to be com pone nts of th

p l a s m a m e m b r a n e 23), t h e P s E N O D 5 p r o te i n m a y b e p ar t o f t h

p lasma mem brane of the infec t ion thread .

Ex p r e s si o n o f t h e Ps E NO D1 2 g e n e i s o b s e r v ed i n r o o t h a i r s a n

in cor t ica l ce ll s tha t conta in a grow ing infec t ion thread . I n cont ra

t o t h e Ps ENOD5 g e n e , t h e Ps ENOD1 2 g e n e i s a l s o e x p r e s s e d i

cells tha t occ ur several layers in front of the gr ow ing in fectio

threads (Fig . 3 , A and B) and a re undergoing the morphologic

changes tha t precede pene t ra t ion by an infec t ion thread 22). T h

p u t a ti v e c el l w a ll p r o t e i n P s EN OD 1 2 m a y t h u s b e p a r t o f th

addit ional cell wall formed in the cortex ceils that prepare fo

infec t ion thread passage . In addi t ion , the PsENOD12 pro te in ma

be a com pon ent of the infec t ion thread i t sel f.

Another p lan t pro te in for which an involvement in the infec t io

process has been demonstrated is lectin.

R. leguminosar ium

biov

(bv.) viciae normal ly infec ts and nodula tes pea and ve tch , bu t

n o

wh ite clover. In part ial ex ception t o the b arriers of host specifici ty

R. leguminosarum

bv.

viciae

i s ab le to induce root ha i r cur l ing o

whi te c lover roots , a l though infec t ion threads a re never formed

Ho wev er , in t rodu ct ion o f a pea lec t in g ene in t ransformed ha i r

roots on whi te c lover a l lows the infec t ion to proceed beyond roo

hai r cur l ing 24). Apparent ly , pea lec tin i s in some way involved

spec i fy ing the hos t spec i f ic i ty of in fec t ion thread format ion . Th

plant receptor of NodR m-l ik e molecules has been pos tu la ted to b

a lectin 18) , a l though exper imenta l da ta have not ye t been pu

forward .

T h e R hiz ob ium genes invo lved in gener ation o f signals tha t rela

to infec t ion thread format ion and growth wi l l be more d i f f icu l t t

ident i fy than the genes and fac tors involved in root ha i r deforma

t ion . For ins tance , gene t ic ana lys i s has ind ica ted tha t the commo

nod genes a re required for in fec t ion thread format ion . H owe ver , th

requi remen t m ay ac tua l ly be for root ha i r cur l ing , which wou ld the

a l low infec t ion thread format ion as a secondary response . Becaus

no nodular s t ruc ture has ever been descr ibed in which an infec t io

thread o r a thread-l ike s t ruc ture i s formed in the absence of th

bac te r ium, the phys ica l presence of R hiz ob ium may be essential fo

infec t ion thread format ion to occur and proceed . Poss ib ly th

d iv id ing bac te r ia a re the dr iv ing force for in fec t ion thread growth

App lication o f steri le culture f i l trates o f R. leguminosarum bv. vici

to pea seedl ings e l ic i t s PsENOD12 gene express ion in root ha i r s

The express ion o f th i s in fec tion process - re la ted p lan t gene show

tha t such a gene can be induced by so luble fac tors wi thout ac tua

infec t ion thread format ion . Appl ica t ion of the pur i f ied ex trace l lu la

fac tor , No dR lv , a l so caused root ha i r deform at ion (2 .5). Al th oug

the s t ruc ture o f th i s No dR lv fac tor has no t been e luc ida ted ye t , it

l ikely to resemble NodRm-1 in several features, as discussed above

Apparent ly , the o l igosacchar ide tha t i s respons ib le for root ha

deform at ion i s al so involved in t r igger ing a t l eas t par t o f th

infection process.

I n a d d i t i o n t o t h e nod genes, genetic analyses of R hiz ob iu

mutants have identif ied numerous other bacterial genes that ar

r e q u ir e d f o r n o r m a l n o d u l e d e v e l o p m e n t 8, 12) . As a rule, rhizob i

muta ted in these genes can induce p lan ts to form nodule- l ik

s t ruc tures , bu t these fa i l to form infec t ion threads , form infec t io

threads th at ab ort prem aturely , or are defective in bacterial releas

f rom the infec t ion threads . Examples inc lude the genes involved

the prod uct io n of bacte r ia l ou te r sur face com pone nts such

exopolysacchar ides

exo

genes) , l ipopolysaccharides

lps

genes) , an

950 SCIENC E VOL. 25

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(cyc l i c ) g lucans

ndv

g e n e s ) , i n a d d i t i o n t o g e n e s r e l a t e d t o d r u g

r e s i s t a n c e , a u x o t r o p h y , a n d c a r b o h y d r a t e m e t a b o l i s m

8, 12, 26).

G e n e t i c e v i d e n c e i n d i c a t e s t h a t t h e

R h i z o b i u m

g e n e s d e s c r i b e d

a b o v e a r e a l l e s s e n t ia l f o r p r o p e r i n f e c t i o n th r e a d f o r m a t i o n , a n d

e a c h o f t h e m m a y a n d o f t e n h a s b e e n c l a i m e d t o p r o d u c e a s ig n a l

t h a t i n d u c e s a s t e p i n t h i s p r o c e s s . H o w e v e r , i n o u r v i e w a t l e a s t

t h r e e d i f f e r e n t c la s s es o f g e n e s s h o u l d b e r e c o g n i z e d i n t h e i n t er a c -

t i o n s b e t w e e n

R h i z o b i u m

a n d t h e l e g u m e p l a n t .

T h e f i rs t c la s s o f g e n e s c o n t r i b u t e s t o t h e s y n t h e s is o f s i g n a l s t h a t

i n d u c e d e v e l o p m e n t a l p r o c e s s e s in t h e p l a n t , a s d e m o n s t r a t e d f o r t h e

nod

g e n e s i n t h e p r o d u c t i o n o f N o d R m - l i k e f a c t or s

18, 19) .

T h e

s e c o n d c l a s s o f g e n e s i s c o n c e r n e d w i t h g e n e p r o d u c t s t h a t a r e

r e q u i r e d f o r i n f e c t i o n , b u t d o n o t i n d u c e a d e v e l o p m e n t a l p r o c es s in

t h e p l a n t . . A n e x a m p l e i s t h e s y m b i o t i c d e f i c ie n c y o f a u x o t r o p h i c

m u t a n t s ( 2 6 ) , w h i c h i n d i c a t e s t h a t t h e b a c t e r i a n e e d t o b e m e t a b o l -

i c a ll y a c t iv e a n d m u l t i p l y i n o r d e r t o i n f e ct . T h e g e n e s p r o d u c i n g t h e

r h i z o b i a l c o m p o u n d s i n t h e m a t r i x o f t h e i n f e c t io n t h r e a d a r e a l s o

l i ke ly t o be lo ng t o t h i s c l ass o f gene s . The t h i rd c lass o f genes i s

i n v o l v e d in t h e d i s g u i s e

o f R h i z o b i u m

i n o r d e r f o r

R h i z o b i u m

t o a v o i d

t h e p l a n t d e f e n s e m e c h a n i s m s a n d t h e p l a n t - b a c t e r i u m i n t e r a c t i o n t o

resu l t in a f i anc ti ona l roo t nod ul e . P l an t s a re ab l e t o defe nd

t h e m s e l v e s a g a i n s t p a t h o g e n s b y a v a r i e ty o f m e a n s ( 8 ) , n o n e o f

w h i c h i s o b se r v e d d u r i n g n o d u l e d e v e l o p m e n t . A p e r t u r b a t io n o f

t h e n o r m a l s i t u a t i o n , h o w e v e r , a p p e a r s t o e l i c it a d e f e n s e r e s p o n s e

( 8 ) . I n o u r o p i n i o n , t h e p r o d u c t s o f t h e

exo, lps,

a n d

ndv

g e n e s m o s t

l ik e l y a c t a s s o - c a l le d a v o i d a n c e d e t e r m i n a n t s t o m a k e

Rh iz o b iu m a

p a r a s i te in d i s g u i s e . A n y m u t a t i o n t h a t u n m a s k s a n a v o i d a n c e

d e t e r m i n a n t w i l l t r i g g e r t h e p l a n t ' s d e f e n s e r e s p o n s e s a n d r e s u l t in

a b o r t i o n o f n o d u l e d e v e l o p m e n t , e v e n i f al l s i g n a ls f o r p r o p e r

d e v e l o p m e n t a r e p r e s e n t .

T h e o n l y b a c t e r i a l g e n e s f o r w h i c h a d i r e c t i n v o l v e m e n t i n

g e n e r a t i n g a s i g n a l t h a t t r i g g e r s d e v e l o p m e n t h a s b e e n d e m o n -

s t ra t ed a re t he

nod

g e n e s , w h i c h p r o d u c e t h e N o d f a c t o r s . Sa t i s f a c -

t o r y e v i d e n c e h a s y e t t o b e p r e s e n t e d t h a t a n y b a c t e r i a l g c n e o t h e r

t h a n t h e

nod

g e n e s i s i n v o l v e d in t h e p r o d u c t i o n o f t h e s i g n al s

e l i c it i n g i n f e c t io n t h r e a d f o r m a t i o n a n d g r o w t h .

P r i m o r d iu m F o r m a t i o n

T h e t h i r d v i s i b l e p r o c e s s i n d u c e d b y r h i z o b i a i s m i t o t i c a c t iv i t y i n

t h e r o o t c o r t e x

6, 14,

2 7 ) . W h i l e t h e i n f e c t io n t h r e a d s p e n e t r a t e t h e

roo t ce l ls and m ove i nwa rd , f ia ll y d i f fe ren t i a t ed ce l l s i n t he r oo t

c o r t e x a r e r e a c ti v a t e d a n d s t a r t t o d i v i d e , t h u s f o r m i n g t h e n o d u l e

p r i m o r d i u m ( 2 , 1 6 ) ( F i g . 3 H ) . W h i c h r o o t c o r t e x c e ll s d i v i d e

d e p e n d s u p o n t h e t y p e o f n o d u l e a p a r t i c u l a r h o s t p l a n t f o rm s . I n

genera l , t empera t e l egumes , such as pea , ve t ch , c l over , and a l fa l fa ,

f o r m i n d e t e r m i n a t e n o d u l e s t h a t a r e c l u b - s h a p e d a n d h a v e a p e rs i s t-

e n t a p i c a l m e r i s t e m . I n t h e s e l e g u m e s , t h e n o d u l e o r i g i n a t e s f r o m

c el ls l o c a t e d i n t h e i n n e r c o r t e x o f t h e r o o t , i n m o s t c a s es o p p o s i t e a

x y l e m p o l e o f th e r o o t v a s c u l a r s y s t e m ( 2 7) ( F i g . 3 F ) . I n c o n t r a s t ,

m o s t t r o p i c a l l e g u m e s , s u c h a s s o y b e a n a n d F r e n c h b e a n , f o r m

g l o b o s e - s t r u c t u r e d d e t e r m i n a t e n o d u l e s , i n w h i c h m i t o t i c a c t i v i t y

c e a se s d u r i n g d e v e l o p m e n t a n d c e ll e x p a n s i o n r a t h e r t h a n c e ll

d i v i s io n i s r e s p o n s i b l e f o r t h e i n c r e a s e in n o d u l e s i ze . D e t e r m i n a t e

n o d u l e s o r i g i n a t e f r o m m i t o t i c a c t i v i ty in d u c e d i n t h e r o o t o u t e r

cor t ex (27 ) .

I n t h e d e v e l o p m e n t o f b o t h d e t e r m i n a t e a n d i n d e t e r m i n a te

n o d u l e s , t h e i n f e c t i o n t h r e a d s g r o w t o w a r d s t h e n o d u l e p r i m o r d i a .

U p o n c o n t a c t , t h e t h r e a d s b r a n c h a n d p e n e t r a t e t h e c el ls o f th e

c e n t r a l r e g i o n o f th e p r i m o r d i u m . I n t h e s e c e ll s , t h e b a c t e r i a b u d o f f

f r o m t h e t i p s o f t h e i n f e c t i o n t h r e a d s i n t o t h e p l a n t c y t o p l a s m . T h i s

r e l e a s e i s a n e n d o c y t o t i c p r o c e s s i n w h i c h t h e b a c t e r i a b e c o m e

e n c l o s e d b y t h e p e r i b a c t e r o i d m e m b r a n e , w h i c h i s i n i ti a l l y d e r i v e d

1 6 N O V E M B E R 1 9 90

f r o m t h e p l a s m a l e m m a o f th e p l a n t c e ll

28) .

C o n c o m i t a n t l y w i t h b a c t e r ia l r e le a s e , s o m e c e ll l ay e r s a t t h e d

s i te o f t h e n o d u l e p r i m o r d i a d e v e l o p i n t o t h e s m a l l c y t o p l a s m

c e ll s t h a t f o r m t h e m e r i s t e m o f t h e i n d e t e r m i n a t e n o d u l e ( F i g . 3

T h e m e r i s t e m g e n e r a t e s t h e d i f f e r e n t t i s s u e t y p e s o f t h e n o d

W h i l e t h e m e r i s t e m a t i c c e ll s a r e p u s h e d o u t w a r d s , t h e i n f e c

t h r e a d s r e v e r s e t h e i r g r o w t h d i r e c t i o n t o f o l l o w t h e n o d u l e m

s t e m . T h u s , t h e g r o w t h d i r e c t i o n o f th e i n f e c t i o n th r e a d a p p e a

b e c o r r e l a t e d w i t h t h e p o s i t i o n o f m i t o t i c a l l y a c t iv e c e l ls 2 8 ) .

I n s i tu h y b r i d i z a t i o n s tu d i e s s h o w t h a t th e n o d u l e p r i m o r d

IZ ~ ~ ' ~ ~ '~ ~ ~ l,~

i

i - ~ ~ , ~ ~ ~ l ~ , --

Fig. 3. Lig ht micrographs of the localization by in situ hybridi zation

antisense RN A of early nodulin m~ As in pea root nodules at diffe

stages of development. (A to D) Lo calization of Ps EN OD 12 (A and B)

PsENO D5 (C and D) mR NA in cross sections of infected pea roots. (A

C), bright-ficld micrographs corresponding to the dark-field micrograp

(B) and (D), respectively. In the dark-field micrographs, silver g

rcprescnting hybridization are visible as white spots. Th e arrows indicat

t ip of the infcction thread. PsEN OD 5 mR NA (D) i s located in the

containing the infection thread tip and in the ro ot cortex cell just passe

thc infection thread. PsEN OD 12 mR N A (B) is located in root cortex

containing the infection thread as well as in cortex cells in fi 'ont of the thr

(E ro G) Local izat ion of PsEN OD 12 mR NA in a mature pea nodule.

bright-field micrograph o f the corresponding dark-field micrograph in

In (E ) the approximate position of the different developmental zones o

determinate root nodule shown in (F) and (G) is indicated. The PsENO

mR NA is restricted to the invasion zone. (H and I) Bright-field microg

(H ) and the corresponding dark-field micrograph (I) of a transsec

through a pea root containing a nodule primo rdium. At the distal si te o

nodule primordium a meristem (M) is formed. P art of the infection th

that carries

Rh iz o b iu m

from the root surface to the nodule primordiu

indicated with arrowheads. At this stage the primordium starts to diffe

tiate. Th e first cell to differentiate into a no dule parenchyma cell hybrid

with antisense PsEN OD 2 RN A (arrow). Abbreviations: (M ), meris

(IZ), invasion zone; (ESZ), early symbiotic zone; (LSZ) late symb

zone; (SZ), senescent zone; (X), xylem pole of the ro ot vascular bundle;

phloem pole of the root vascular bundle; (VB), vascular bundle of

nodule; (C), central tissue.

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can be d i s tinguished f rom the nodule mer i s tem by molecular c r ite r ia

in addi t ion to i t s d i s t inc t morpho logica l fea tures

22).

The infec tion-

re la ted P sE N OD 12 gene i s expressed in al l cel ls of the pr im ordium ,

w h e r e as Ps EN OD 1 2 m R N A i s n o t d e t e ct a b le i n th e c e ll s o f th e

nodule m er i s tem. Most l ike ly, the express ion of the PsE NO D1 2

gene re f lect s the condi t ion ing of the dedi f fe ren t ia ted pr im ordiu m

cel ls for in fec t ion , bu t Ps EN OD 12 may a lso have a def ined func t ion

i n p r i m o r d i u m f o r m a t i o n .

NodRm-1 appl ied to a l fa l fa seedl ings in approximate ly a hun-

dredfo ld h igher concent ra t ion than requi red for cur l ing induces

mi to t ic ac t iv ity a t s i tes in the inner cor tex where nodule pr im ordiu m

format ion i s normal ly induced by

Rhizobium

(19). As a result o f this

mitotic activity, nodule-l ike structures develop (19). A single bacte-

rial ol igosaecharide is thus able to el ici t a wide range of responses

f rom i t s l egume hos t . The o l igosacchar ide na ture of No dR m- 1 i s

cons is ten t wi th o ther observa t ions tha t o l igosacchar ides can regula te

p lan t morphogenes is

29).

Different ia t ion into

a R o o t N o d u l e

After mer i s tem format ion , d i f fe ren t nodule t i s sues a re formed

from the nodule mer i s tem. In f i f i ly deve loped root nodules , two

major t issue types can be recognized, the central t issue, and the

peripheral t issue (Fig. 3F). Th e peripheral t issue consists of the

nodule cor tex and the nodule endod ermis , in addi t ion to the nodule

parenchym a ( inner cor tex) , which conta ins the vascular bundles tha t

connec t the nodule wi th the root s te le

28).

The t i s sue m ost charac te r is t ic of the root nodule i s the cent ra l

t issue. In the central t issue of the indeterm inate no dule, thre e

developmenta l zones can be d i s t inguished (Fig . 3 , E and F) . In the

invas ion zone immedia te ly ad jacent to the mer i s tem, re lease of

bac te r ia f rom the infec t ion threads cont inues to es tab l i sh newly

infected cells . Approx ima tely ha lf of the cells are not penetr ated by

infec t ion threads and remain uninfec ted . The invas ion zone i s

followed by the early symbiotic zone, in which plant cells elongate

anct bacteria proliferate. In the late symbiotic zo ne, t l- ie infected cells

are completely f i l led with bacteria that have differentiated into their

p le iomorphic endosymbiot ic bac te ro id form. Ni t rogen f ixa t ion

takes place in this late symbiotic zone. In older nodules, a fourth

zone is the senescent zone, in which both plant cells and bacteroids

degenera te . D ue to the pers i stence of the ap ica l mer i s tem, a l l

deve lopmenta l phases of the inde te rmina te nodule a re represented

within a single nodule.

Determina te nodules lack a pers i s ten t mer i s tem. As a conse-

quence , the deve lopment and f ina l organiza t ion of a de te rmina te

nodule differs from the indeter mina te n odule. All cells of the central

t i s sue wi th in a s ingle de te rmina te nodule a re progress ing through

t h e s a m e s t a g e o f d e v e l o p m e n t

28).

The smaller uninfected cells

found interspersed among the infected cells in the central t issue of

determ inate nodules are specialized for the assimilat ion o f f ixed

ni t rogen to ure ides (5) . No evidence has been found for such a

specialization of the unin fected cells in indetermin ate n odules.

Studies on p lan t gene express ion conf i rmed the ex is tence of

severa l d i s t inc t deve lopmenta l zones in an inde te rmina te root n od-

ule

21).

The deve lopm enta l zon es as def ined by cy to logica l c r i te r ia

coincide more or less with the zones characterized by the expression

of par t icu la r ear ly nodul in genes . The ear ly nodul in gene

Ps EN OD 12 i s expressed in a ll ce ll s of the invas ion zone , w hich i s

cons is ten t wi th a f imct ion of PsE NO D1 2 in the infec t ion process

22)

( F ig . 3 G) . I n o l d er p a r ts o f t h e n o du l e , Ps E NO D1 2 m R NA i s

not de tec tab le . The PsENOD5 gene express ion i s apparent in the

invas ion zone of the cent ra l t i s sue , bu t to the grea tes t ex ten t in the

infec ted ce l ls of the ear ly symbiot ic zone

21).

In these cells,

952

in fec t ion threads have s topped growing, bac te r ia pro l i fe ra te , and

very ac t ive membrane-synthes iz ing appara tus sur rounds the pro l

e ra t ing bac te r ia wi th the per ibac te ro id membrane . The re la t ive

high ac t iv i ty of the PsENOD5 gene in these ce l l s sugges ts tha t t

a rab inogalactan- l ike pro te in Ps EN OD 5 may not on ly be par t o f t

p lasma me mbra ne of the infec t ion thread , b u t a l so par t o f t

per ibac te ro id membrane .

In the ear ly symbiot ic zone , two o ther ear ly nodul in gene

Ps E NO D3 a n d Ps E NO D1 4 , a r e e x cl u si ve ly e x p re s s ed i n th e

fected cells of the central t issue (21). Sequence analyses of Ps

N O D 3 a n d P s E N O D ] 4 e D N A c lo n es s h o w t h a t t h e m R N A

encode 6-kD polypept ides tha t a re 55% homologous . They have

puta t ive s igna l pept ide a t the amino te rmina l end and conta in fo

cys te ine res idues wi th a spa t ia l d i s t r ibu t ion resembl ing tha t

m e t a l - b i n d i n g p r o t e i n s . T h e Ps ENOD3 a n d Ps ENOD1 4 n o d u l i

may be me ta l -b inding pro te ins involved in , for ins tance , t ranspor t

Fe or Mo to the bac te ro ids which requi re these meta l s for

f ianc t iona l n i t rogen/hdase . Simi la r to the PsENOD12 and Ps

N OD 5 g e n e s, t h e Ps - E NO D3 a n d Ps EN OD 1 4 g e n e s a r e t r an s ie n

expressed dur ing the deve lopm ent of the nodule . In the la

s y m b i o t ic z on e , t h e a m o u n t s o f Ps E NO D3 a n d P .s ENOD 1

mRNAs decrease , and la te nodul in mRNAs, such as for l eghem

globin , become de tec tab le .

Physiological studies have indicated that the peripheral t iss

cont r ibutes to the spec ia l fea tures of root nodule mor pholo gy

3

The c omp act s t ruc ture of the nodule p arenchyma wi th i t s few a

small intracellular spaces forms an oxygen diffusion barrier .

comb ina t ion w i th the h igh oxygen consump t ion ra te of the bac te

and the presence of leghemoglobin , th i s bar r ie r he lps to main ta

the low f ree oxygen concent ra t ion in the cent ral t i s sue as requi r

for n i t rogenase func t ion .

The specia l charac te ri s t ics of the nodule parenchym a are a l

reflected in the pattern of early nodulin gene expression. The ear

nodul in gene P sE NO D2 i s speci f ica l ly expressed in the nodu

p a r e n c h y m a o f p e a

31).

Si m il a rl y , G m E N OD 2 g e n e e x p re s s io n

the de te rmina te soybean nodule i s conf ined to the nodule pare

c h y m a . P s E N O D 2

31)

a n d G m E N O D 2

32)

are structura

s i m il a r t o P s EN OD 1 2 , i n th a t b o t h a r e m a i n l y c o m p o s e d o f t w

al te rna t ing pentapept ides , each of which conta ins two pro l i

residues. This structure suggests that these early nodulins are c

wal l comp onents . Because the ce ll wal l i s the majo r de te rminant

ce l l morphology, i t i s l ike ly tha t an ear ly nodul in such as ENOD

cont r ibutes to the spec ia l morphology of nodule parenchyma ce l l

which i s requi red for es tab l i sh ing the oxygen d i f fus ion bar r ier .

N o d u l e F unc t i on i ng

The es tab l i shment of the ear ly symbiot ic zone marks the end

the morp hologica l changes acc ompanying d i f fe ren t ia t ion in to a ro

nodule . Next , b iochemica l a l te ra t ions brought about by , amo

other th ings , l a te nodul in gene express ion , c rea te and suppor t t

requi red envi ronment for n i t rogen f ixa t ion and ammonia ass imi l

t ion to occur . La te nodul in gene express ion has been demonst ra t

in a large variety of legume species

8, 10) .

Nu m e r o u s l at e n o d ul

genes , inc luding a l l soybean leghemoglobin (Lb) genes , have be

sequenced and a l ist of al l cloned (late) nodulin genes has be

presented recent ly

33).

The bes t s tud ied la te nodul in a l so i s the most abundant one : L

cons t i tu tes up to 25 % of the to ta l so luble pro te in in a matu

nodule . Lb i s an oxyhemoprote in wi th a h igh oxygen af f in i ty

3

and resembles the ver tebra te g lobins . In combina t ion wi th t

spec ia l nodule m orpho logy, L b cont ro ls the concent ra t ion o f f

oxygen in the nodule . I t p rovides a f low of oxygen toward t

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bac tero ids tha t ba lances pro tec t ion o f n i trogenase aga ins t oxygen

damage wi th suppor t of resp i ra t ion . Lb may be a t rue "symbiot ic

pro te in , " because the heme group i s presumed to be a bac te ro id

product , whereas the g lobin par t i s encoded by the p lan t genome

(8).

Othe r la te nodul ins have been ident if ied as enzymes or subuni t s o f

enzymes tha t func t ion in n i t rogen (g lu tamine synthetase , u r icase) or

carbon (sucrose synthase) metabolism (8). These late nodulins al l

per form tasks tha t ind iv idua l ly a re not un ique to nodule func t ion-

ing, but occ ur in other p arts of the plant as well. Based on this and

other ob serva t ions not to be d i scussed here , we have put forward the

hypothes i s tha t nodule format ion evolved f rom re la t ive ly minor

a l te ra t ions in the pa thw ay of root d i f fe ren tia t ion , in which com mo n

plant genes: becam e adapte d to f i t the physiological or reg ulatory

cons t ra in ts o f the symbios i s (35) . Several nodule enzym es have been

shown to differ in physical , kinetic, or immunological characterist ics

f rom the forms found in roots (36) : phosphoeno lpyruvate carboxyl -

ase, choline kinase, xanthine dehydrogenase, purine nucleosidase,

and mala te dehydrogenase . I t i s unknown whether the nodule-

spec i f ic forms of these enzymes or ig ina te f rom the express ion of

nodulin genes, or result from nodule-specific, post- translat ional

modi f ica t ions of gene prod ucts a l so found e l sewhere in the p lan t .

La te nodul ins a re a l so assoc ia ted wi th the per ibac te ro id mem-

brane (8) . This membrane enc loses the bac te r ia wi th in the p lan t

cy toplasm, thus forming the phys ica l and metabol ic in te r face be-

tween bac te r ium and p lan t . The prec ise b iochemica l func t ion of

most o f these per ibac te ro id mem brane nodul ins i s as ye t unknow n.

The bes t s tud ied i s the soybean nodul in Ngm-26, which probably i s

a t ransmem brane pro te in spanning the fu ll per ibac te ro id mem brane .

I t s amino ac id sequence i s hom ologo us to the

Escherichia co li

glycerol

fac i li t a tor pro te in (37) , which i s the only known pore- typ e pro te in

in the E. col i cytoplasmnic membrane and which functions in trans-

por t o f smal l molecules . Rece nt resu l ts demons t ra te tha t N gm -26

forms an ion channel l ikely to function in translocation of small

compounds , l ike the carbon source succ ina te , across the per ibac-

t e r o i d m e m b r a n e 38) .

All la te nodul in genes app ear to be coord ina te ly expressed in t ime

and development (8, 10). These genes might thus al l be activated as

a result of a single signal , perhaps related to release of bacteria from

the infec t ion threads , because they a re not expressed in nodules

wi thou t in fec ted cel ls . The n a ture o f th i s presumed s igna l , however ,

and the involvement of bac te r ia l genes remain unc lear .

T h e i m p o r t a n c e o f t h e p r o m o t e r r e g i o n o f n o d u l i n g e n e s f o r

nodule-spec if ic gene express ion has been dem onst ra ted wi th the use

o f t r an s g e n ic p l a n ts . T h e p r o m o t e r r e g io n o f a s o y b e a n L b g e n e

di rec ted nodule-spec i fic and deve lopmenta l ly cor rec t express ion o f a

repor te r gene when t ransformed in to o ther leguminous p lan ts . Ap-

parent ly , the Lb promoter reg ion not on ly car r ies a l l requi red cis-

regulatory sequences, but al l relevant trans-acting factors are also

conserved among different leguminous species. Deletion analysis of

the soybean Lb promoter defines a relat ively small region as respon-

sible for the correct expression patrtem 39) . In soybean , a nodule-

specific

tram-acting

fac tor in te rac t ing wi th the Lb promoter has been

identified 40) and awaits cloning. Molecular analyses of promoters

and their tram-acting

factors wil l give insight in the mechanisms of

nodt fl in gene regula t ion and may h in t to the na ture and or ig in o f the

signal presumed to tr igger the expression of these genes.

In si tu hybridization studies with the use of bacterial antisense

RN A showed tha t the rh izobia l n / f genes , encoding n i t rogenase ,

were expressed later than the late nodulin genes 41). This conf i rmed

the not ion tha t the nodule prepares for n i t rogen f ixa t ion by synthe-

s iz ing la te nodul ins . I t a l so sugges ted ye t another s tep in the

communica t ion be tween p lan t and bac te r ium to y ie ld symbiot ic

nitrogen fixation.

1 6 N O V E M B E R 1 9 90

Concluding Remarks

Appl ica t ion o f No dR m-1 to legume roots i s suf fic ien t to ind

root ha i r defo rmat ion , express ion of the ear ly nodul in g

Ps EN O D 12 ( representa t ive of a t l eas t par t o f the infec tion proce

and mitotic activitT in the root cor tex fo l lowed by format ion o

nodule-l ike structure 19) . NodRm-l ike fac tors thus have a p ivo

role in al l three processes induced by R hiz ob ium (Fig. 1) .

o l igosacchar ide na ture o f the N odR m-1 fac tor i s cons is ten t w

observa t ions tha t o l igosacchar ides can regula te p lan t morphogen e

29).

The na ture o f the s igna ls involved in induct ion o f la te nodu

gene express ion i s no t known , b ut the presence of in fec tion thre

or intracellular bacteria appears to be required. Therefore,

signals tr iggering late nodulin gene expression are most l ik

genera ted by the re lease of the bac te ria f rom the infec tion threa

I t i s cur ren t ly an open ques t ion how a s ingle fac tor can ind

three apparently different processes. I t might be possible that ,

example, mitotic activity in the root cortex is induced by a seco

s ignal genera ted in deformed o r cur led root ha i r s 42). Alternative

the different processes induced by

R hiz ob ium

might share cer t

aspec ts . Infec t ion and induct ion of mi to t ic ac t iv i ty seem ra t

d i f feren t , bu t a re la tionship be tween both fo l lows f rom the hypo

esis that cells preparing for infection thread growth begin divis

and a re arrested in the pro phas e o f the cell cycle (14).

Another poss ib i l i ty might be tha t the Nod fac tor induces

d i f fe ren t deve lopmenta l processes , bu t p lan t fac tors de te rmine

outco me of deve lopment . An involvement of p lan t fac tors in s teps

nodule format ion has been demonst ra ted 43). Root nodules

induced pred omina nt ly oppo s i te a xylem pole (Fig . 3F) , and

alcoholic extract of the root stele is able to influence the pattern

cort ical cell divisions in the root cortex

43).

Therefore , i t has b

suggested that factors from the xylem are involved in tr iggering c

d iv ision . I t i s conce ivable tha t a gradien t o f the Nod fac tor , o r a N

fac tor -der ived s igna l , decreas ing in concent ra t ion f rom infec t

si te root inwards, interacts with a xylem factor gradient in

oppos i te d i rec t ion . This in te rac tion m ay de te rmine whether cor t i

ce l l s d iv ide to form the nodule pr imordium or become ar res ted

the i r ce l l cyc le as prepara t ion for pene t ra t ion by the grow

infection thread.

The mechanism by which a Nod fac tor t r iggers p lan t deve l

men t i s unknow n, but may involve changes in phyto horm one ra t

Add it ion of auxin transp ort inh ibitors to alfalfa roots gives rise

nodule-l ike structures, in which early nodulin genes are expres

44) and which cy to logica l ly resemble R hiz ob ium- induced nodul

Fur the rmore , the in t roduct ion of a zea t in gene, involved in cy to

n i n s yn t h es i s, c a n p a rt l y c o m p l e m e n t N o d - m u t a n t s o f R . mel i

(45) . The No d fac tor may thus accompl i sh changes in the ba lance

phytohormones in the root , which secondar i ly (46) resu l t in nod

format ion .

T o u n d e r s t an d m o r e a b o u t t h e m e c h a n i s m o f No d f a c to r a c ti

the way the s igna l g iven by the Nod fac tor i s perce ived a

t ransduced by the p lan t m us t be e luc ida ted . Ident i f ica t ion o f

plant receptors involved, as well as research into the signal tra

duc t ion pa thways in the legume p lan t , wil l be fac il i ta ted enormou

by the oppor tu ni t ies to obta in subs tan t ia l amounts o f pur i fied N

factors from bacterial cultures.

The poss ib le involvement of phytohormones in qodt , lc deve l

ment sugges ts tha t the prokaryote R hiz ob ium has acquired the abi

to cope wi th the endogenous p lan t sys tems tha t regula te deve l

ment . In te rms o f symbios is , i t would seem to make sense tha t

bacterium employs those plant systems as much as t~)ssiblc. T

cha l lenge no w i s to d i scover what the bac te r ium apparent ly a l re

know s. Fu rther e xploitat ion o f this prokaryo tc and i ts signals

cont r ibute to our und ers tanding of the deve lopmenta l b io logy

A R T I C L E S

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roo t nodu le fo rmat ion , and be o f general sign if icance fo r our ins igh t

in to the development o f p lan ts .

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47. We than k H. J. Franssen, F. Govers , B, Horvath, and A. van Kam men for cri t ic

reading of the manuscript , M . J. va n Iersel , G. H ei tk6nig, and L. Kesaulya-Monst

for secretarial ass is tance, P. Madem for drawings, and C. van de Wiel f

cytological help in preparing Fig. 3.

M o l e c u l a r C h a p e r o n e s : T h e P l a n t C o n n e c t i o n

R .

JOHN

ELLIS

M o l e c u l a r c h a p e r o n e s a r e a f a m i l y o f u n r e l a t e d p r o t e i n s

f o u n d i n a l l t y p e s o f c e l l . T h e y m e d i a t e t h e c o r r e c t

a s s e m b l y o f o th e r p o ly p e p f i d e s b u t ar e n o t c o m p o n e n t s

o f t h e m a t u r e a s s e m b l e d s t n a c tu r e s . C h a p e r o n e s f u n c t i o n

b y b i n d i n g s p e c if i ca l ly t o i n t e ra c t iv e p r o t e i n s u r f a c es t h a t

a r e e x p o s e d t r a n s i e n t l y d u r i n g m a n y c e l l u l a r p r o c e s s e s

a n d s o p r e v e n t t h e m f r o m u n d e r g o i n g i n c o rr e ct i n t e r ac -

t i o n s t h a t m i g h t p r o d u c e n o n f i m c t i o n a l s t r u c t u r e s . T h e

c o n c e p t o f m o l e c u l a r c h a p e r o n e s o r i g i n a t e d l a r g e l y f r o m

s t u d i e s o f t h e ch l o r o p l a s t e n z y m e r u b i s co w h i c h f i x e s

c a r b o n d i o x i d e i n p l a n t p h o t o s y n t h e s i s ; t h e f i a n c t i o n o f

c h a p e r o n e s f o r c e s a r e t h i n k i n g o f t h e p r i n c ip l e o f p r o t e i n

se l f -a sse mbly .

T

H E

S T U D Y O F T H E M O L E C U L A R B I O L O G Y O F P L A N T S IS

undergo ing a rap id expansion , fueled bo th by techn ical

advances and by the real izat ion that there may be ec onomic

advan tages i f p lan ts can be man ipu lated in new ways. One o f the few

basic concep ts in molecu lar b io logy that has o r ig inated f rom

research wi th p lan ts is that o f molecu lar chaperones . Th is con cep t

9 5 4

developed f rom stud ies on the b iogenesis o f the ch lo rop las t enzym

that f ixes carbon d iox ide in pho tosyn thesis ( r ibu lose b isphosphat

carboxy lase-oxygenase o r rub isco) , bu t the f ie ld now encompasse

animal and microbial cells and has medical and biotechnologic

aspects (1 -10) . In th is ar t ic le I descr ibe how the c haperone con cep

developed f rom stud ies on rub isco and d iscuss the impl icat ions o

th is concep t f o r fu tu re p lan t research .

Rubisco Biogenesis

Rubisco ind irectly o r d i rect ly -p lays a v i ta l ro le in the metabof ism

of all cells, since it is the principal catalyst that brings carbon int

o rgan ic combinat ion f rom atmospher ic carbon d iox ide dur ing pho

tosyn thesis . I t s b iogenesis i s unusual ly complex and invo lves th

in teract ion o f l igh t as a developmental t r igger wi th tw o d is t inc

genet ic systems, one located wi th in the ch lo rop las t and the o the

with in the nucleus (11) . Desp i te i t s v i ta l ro le , rub isco is a poo

catalyst and has bo th a low aff in i ty fo r carbon d iox ide and a sma

tu rnover number; thus au to t roph ic o rgan isms devo te a majo r pa

The author is professor in the Department of Biological Sciences, Universi ty

Warwick , Coven t ry CV4 7AL, Un i t ed Kingdom.

S C I E N C E V O L . 2 5

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