Legume Root Response to Symbiotic Infection.
Enzymes of the Peribacteroid Space
R obert B. Mellor, E rhard M örschel, and D ietrich W erner
Fachbereich Biologie, Botanisches Institut der Philipps-U niversität, Lahnberge, D-3550 Marburg-L, FRG
Z. Naturforsch. 39 c, 123 - 125 (1984); received N ovem ber 2, 1983
Glycine m ax, Mannosidase, Peribacteroid Space. Rhizobium
Three of the major soluble partitioned pools in the sym biotic cell, namely the host cell
cytoplasm, the cytoplasm of the bacterial symbiont and the space between the two partners
(the peribacteroid space) were assayed for selected enzymes. W hilst alanine dehydrogenase was
found almost totally in the bacterial cell cytoplasm, other enzymes were more widely spread.
Amongst these a-mannosidase, previously described as a vacuolar m arker enzyme in eukaryotes,
was found largely in the peribacteroid space.
These results are discussed in relation to models o f peribacteroid m em brane biogenesis.
Introduction
D uring the initial invasion o f plant root cells by
symbiotic Rhizobium the m em brane which surrounds
the prokaryote is initially do n ated by the plant
plasm a m em brane [ 1 ] but it is unclear as to w hether
the plasm a m em brane continues to con trib u te
m aterial during the subsequent m assive p ro lifera­
tion o f these m em branes. In m any ineffective
(non-N^-fixing) sym bioses the p eribacteroid m e m ­
brane has a m uch shorter life tim e than in effective
(N 2 -fixing) sym bioses. It may be therefore th at the
form ation and m aintenance o f these organelle-like
structures is im p o rtan t in the establishm ent and
m aintenance o f a properly regulated sym biosis.
Results and Discussion
A ntiserum was raised against pure a-m annosidase
from jack bean w hich gave quan tity dep en d en t
precipitate heights w hen challenged w ith antigen in
a L aurell-type “ rocket” im m unoelectrophoresis
system (Fig. la ). A ntigenic cross-reactivity w ith
au thentic Glycine may nodule a-m annosidase was
established sim ply by putting nodule 50000 x g
supernatent into a well in the agar punched closer
than norm al to one containing the original antigen.
The enzymes w ere allow ed to diffuse overnight
before electrophoresis as norm al the next m orning.
The fusion o f the two rockets indicates a com m on
antigenic identity (Fig. 1 c).
Reprint requests to D. W'erner.
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Fig. 1. Immunoelectrophoresis into agarose gels containing
200 jal IgG per plate. Lanes 1 -3 and 8 contain authentic
jack bean a-mannosidase and lane 9 contains authentic
soybean a-mannosidase (Plate C is magnified x l3 ).
Lane 4: 20 ng peribacteroid membranes. 5: 100 |ig bacteroids with peribacteroid membranes intact. 6 : 5 ug p eri­
bacteroid space. 7: 100 jag host cell cytoplasm.
Bacteroids both retain in g and devoid o f su r­
rounding p eribacteroid m em b ran es w ere p rep a re d
as before [2]. From this fraction an osm otic shock
fraction, containing the m olecules present w ithin the
peribacteroid space, the p erib actero id m em b ran es
and also the released bactero id s w ere p u rified [2], It
can be seen from the activities o f various enzym es
(Table I) that som e enzym es were asym m etrically
distributed am ongst these fractions. In p articu la r
alanine dehydrogenase, a bacterial cytoplasm ic
marker, was found overw helm ingly in the 50000 x g
supernatent from F rench-pressed b actero id s w ith
little appearing in then nodule cytoplasm or peribacteroid space fractions, indicating m in o r b actero id
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124
R. B. Mellor et al. • Legume Root Response to Infection
breakage upon hom ogenization. C onversely the
nodule cytoplasm ic m arker enzym e, asp arag in e
synthetase, was confined to this fraction alone,
dem onstrating that little, if any, cytoplasm ad h e red
to the outer surface o f the p erib actero id m e m ­
branes. This view is reenforced by electron m ic ro ­
graphs (Fig. 2) of the bacteroid and p erib actero id
m em brane fraction, show ing no discernable cyto-
Table I. % distribution of soluble enzymes in sym biotic cell
fractions.
Host
cytoplasm
alanine dehydrogenase
asparagine synthetase
x-galactosidase
x-elucosidase
a-mannosidasea
protease b
protein
11.40
100
100
74.73
56.5
35.00
96.52
Peri­
bacteroid
space
0
0
0
9.27
43.48
17.50
1.42
Bacterial
cytoplasm
88.60
0
0
16.0
0
47.60
2.12
a 100% Activity here corresponds to 23% of the total
cellular activity, 77% being recovered as particulate enzyme.
b Measured as PNP release from PNP phenylalanine.
2a
plasm ic contam ination. It thus appears likely th at
the peribacteroid space fraction used was largely
free o f plant or bacteroid-derived soluble c o m p o ­
nents.
By a com parison o f the heights o f th e rockets
formed from standard antigen w ith th o se o f
authentic sam ples (Fig. 1 b) calculations could be
m ade concerning the concentration o f a-m an n o sidase in each sample. As is show n in Fig. 1,
preparations o f whole b acteroids with su rro u n d in g
mem branes, when p rein cu b ated with 0.5% T rito n
X-100, showed a discernable rocket upon im m u n o electrophoresis, w hereas sim ilarity treated p u rified
peribacteroid m em branes did not. It is very unlikely
that any of this a-m annosidase cam e from the
prokaryote, as previous studies [3] and o u r ow n
unpublished im m unoreactive studies show a total
absence o f this enzyme in bacteroids or free-liv in g
cells of Rhizobium japonicum strain 61-A -101.
F urtherm ore alm ost all o f th at activity could be
accounted for w ithin the perib actero id space frac­
tion which gave a p ro m in en t rocket p re c ip ita te
upon im m unoelectrophoresis. Since, on a p ro tein
2b
Fig. 2. Electron micrographs of the fraction designated bacteroids (B) and bacteroids with intact peribacteroid m em ­
branes (P). a: magnification x21 000 (Bar = 0.5 (am); b: m agnification x 57 000 (Bar = 0.2 |im).
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R. B. Mellor et al. • Legume Root Response to Infection
basis, the concentration o f x-m annosidase was som e
15 tim es higher in the peribacteroid space fraction
than in the nodule cytoplasm it appears clear th a t
the space betw een the bacteroids contains x-m annosidase which cannot be accounted for by co n tam in a­
tion.
If we assum e that com ponents o f organelles and
their m atrices are provided by the ER, as is cu r­
rently thought [4] then we w ould expect x-m annosidase, a vacuolar m arker enzym e in yeast and
higher plants [5, 6 ] to be m ade by rough m icrosomes. In infected cells o f soybean nodules vacuoles
never fully develop. M ature sym biotic cells show no
central vacuole as in norm al root cells, and only
fragm ents o f tonoplasts are som etim es faintly
observable [1, 7] but x-m annosidase enjoys an
activity cycle independent o f central vacuole
developm ent [3]. It is know n th at x-m annosidase
exhibits a relatively w ide particulate d istrib u tio n on
this tissue [3] and we have previously speculated
that although vacuole developm ent and m a in ­
tenance is shut dow n in infected tissue, the enzym e
x-m annosidase is still m ade b u t is rerouted else­
where. How then does this m olecule get into p e ri­
bacteroid spaces? If the p eribacteroid m em brane is
form ed from plasm am em brane, m icrobodies w ould
be expected to be form ed from either ER or G olgi,
but first fuse w ith the plasm a m em brane, releasing
their contents outside the cell. Such m icrobodies are
rare [ 1 ] and furtherm ore a m em brane recycling
model now dem ands the selective reup tak e o f
x-m annosidase into nascent peribacteroidal struc­
tures. We know o f no evidence to support these
dictates. Conversely a m em brane recycling m odel
would w ork if x-m annosidase was m ade on cyto­
plasm ic polyribosom es and later specifically tran s­
ported across the peribacteroid m em brane and into
[1] J. W. Kijne, Physiol. Plant Pathol. 5,75 (1975).
[2] J. G. Robertson, M. P. W arburton, P. Lyttleton, A. M.
Fordyce, and S. Bullivant, J. Cell Sei. 30, 151 (1978).
[3] R. B. Mellor, W. Dittrich, and D. Werner, Physiol.
Plant Pathol, (in the press) (1983).
[4] G. Palade, Science 189, 347 (1975).
[5] A Wiemken, M. Schnellenberg, and K. Urech, Arch.
Microbiol. 123,23 (1979).
[6 ] R. A. Leigh and D. Branton, Plant Physiol. 58, 656
(1976).
[7] D. Werner and E. Mörschel, Planta 1 4 1 ,169 (1978).
[8 ] R. B. Mellor, P. Rowell, and W. D. P. Stewart,
Lectins, Biology, Biochemistry and Clinical Bio­
125
the peribacteroid space by a h ith e rto un elu cid ated
mechanism.
We m ust conclude th erefo re from the d ata
presented that p erib actero id m em b ran e p ro lifera­
tion is supported directly by the b iosynthetic parts
o f the endom em brane system in co n tin u ity w ith the
vacuom e in a fashion in d ep en d en t o f the plasm a
m em brane.
Acknowledgement
We thank the D eutsche F o rschungsgem einschaft
for financial support.
Experimental
Glycine max var. m an d arin was su p p lied by
K. Behm G m bH . H am burg and Rhizobium japonicum strain 61-A-101 was from the A m erican Type
Culture Collection. Tissue was grow n and p rep ared
as previously [3].
Cell fractionation was accom plished by follow ing
the scheme detailed in [2 ],
Samples for electron m icroscopy w ere p rep ared
as in [2] and exam ined as in [7],
A ntiserum was raised as before [8 ] in m ale
D eutsche G ro ß silb er rab b its using x-m an n o sid ase
isolated com m ercially from ja ck b e an (Sigm a, M ü n ­
chen) as antigen. Im m u n o electro p h o resis was p e r­
form ed using an LKB M u ltip h o r kit and m ethods
recom m ended by the m a n u fa ctu re r (LKB, U p p sala,
Sweden).
Estim ations o f lytic enzym e activity was as in [9],
alanine dehydrogenase was assayed as by M üller
and W erner [10] and asp arag in e synthetase as
/y-aspartylhydroxam ate fo rm atio n follow ing the
m ethod o f Ravel [11].
Protein d eterm in atio n [12] was carried o ut on
8 % TCA precipitated m aterial.
[9]
[10]
[11]
[12]
chemistry (T. C. Bog Hansen, ed.), pp. 105-112, W.
de Gruyter, Berlin 1982.
S. Bassarab, W. Dittrich, R. B. Mellor, and
D. Werner, Physiol. Plant Pathol, (in the press)
(1983).
P. Müller and D. W erner, Z. N aturforsch. 37 c, 927
(1982).
J. M. Ravel, Meth. Enzymol. 17 A, 722 (1970).
O. H. Lowry, N. J. Rosebrough, A. L. Farr, and
R. J. Randall, J. Biol. Chem. 193,265 (1951).
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