Auzac, J. d'

Chrestin, Hervé

Gidrol, X.

Jacob, J.L.

Marin, Bernard

Horizon / Pleins textes

' i  
Üherreicht vom Viasser . Nicht einzeln im Buchhandel! 
Sonderdruck aifi ~~Zeitschrijì f ir Pf2.?~zenp~~siologie~~, Band 114, Heft 3, S. 269-277 (1984) 
Gustav Fischer Verlag Stuttgart 
Role of the Lutoidic Tonoplast in the Control of the 
Cytosolic Homeostasis within the Laticiferous Cells of 
Hevea 
H. CHRESTIN"), x.GIDROL, B. MARIN'), J. L.  JACOB^) and J. D'AUZAC~) 
') Laboratory of Plant Physiology; O.R.S.T.O.M.; B.P. V51; Abidjan, Ivory Coast 
') Laboratory of Plant Physiology; 1.R.C.AJG.E.R.D.A.T.; B.P. 5035; 34 032 Montpellier 
') Laboratory of Applied Plant Physiology, U.S.T.L. Place E. Bataillon, 34 060 Montpellier, 
Cedex, France 
France 
Received September 13, 1983 . Accepted December 21, 1983 
Summary 
Lutoids, the vacuo-lysosomes of the Hevea latex cells, compartmentalize, in vivo, numerous 
ions such as H', Mg++, Ca", Pi, citrate, some of them strongly toxic for the cytosolic metab- 
olism. Evidence is given for the correlation of the in vivo compartimentation of some of these 
ions inside the lutoids with the latex production by Hevea. 
Two opposing H+ pumps were localized on the lutoidic tonoplast; the one is a Mg'+-de- 
pendent ATPase, the other a NADH-consuming redox system (cytochrome c: artificial ac- 
ceptor). The functioning of these H+ pumps may account for a major part of the transtono- 
plastic ApH variations, and therefore the cytosolic p H  control, which probably regulates the 
highly pH-dependent latex metabolism. 
The resulting proton-motive force energizes the accumulation and compartmentation of 
the inhibiting ions inside the lutoïds, and ensures the control of the <fdetoxificationn and ionic 
equilibrium of the cytoplasm of the laticiferous cells. 
Treatment of Hevea bark with ethrel, an ethylene generator which <<stimulates), latex pro- 
duction, induces an increase in the H'-pumping ATPase activity, resulting in the activation of 
the metabolism in the latex cells. 
All the results reviewed lead us to propose that the lutoids play a double role as a <<bio- 
physical pH-STAT and a <<detoxicating trap,, thus controlling the cytosolic homeostasis. 
Key words: Hevea hasiliensis, laticgen, bitoids, protons pumps, A TPase, NAD(P)H-oxidase, to- 
noplast. 
The latex from Hevea hasiliensis is a fluid cytoplasm which is expelled from 
wounded laticiferous ccvessels,, (articulated, anastomosed cells) (Archer et al., 1963). 
It contains a vacuolar compartment: the so-called <<lutoids,,, consisting of micro-va- 
cuoles with lysosomal characteristics which can be easily isolated and purified by 
simple differential centrifugation (Pujarniscle, 1968; Ribaillier et al., 1971; d'Auzac 
et al., 1982). 
'&) Officially changed name Chréstin Hervé from Crétin Hervé. 
2. Pflalzzenphysiol. Bd. 114. S. 269-277. 1984. I 
Documentaire ORSTOM 
270 H. CHESTIN, X. GIDROL, B. MARIN, J.L. JACOB and J. D’AUZAC 
\ 
6 4  6,5 7,o pH .C 
Fig. 1: Correlation between the cytoplasmic p H  (PH C) and the &toidic>> (vacuolar) pH 
(pH.L) in the freshly collected latex from rubber trees (measured by mean of a pH electrode). 
Table 1: Table of the correlation coefficients linking the latex production (g dry rubbedtap- 
pindtree), the latex cytosolic pH, the transtonoplastic pH gradient, the latex cytosolic and va- 
cuolar citrate concentrations (&) and the resulting transtonoplastisc citrate gradient [data 
obtained from freshly collected latex of 56 rubber trees (+‘bb): very high significance; (**): high 
significance]. 
Latex Vacuolar Cytosolic (Citrate) Cytosolic transtonoplastic 
Production (citrate) (Citrate) gradient p H  p H  gradient 
Latex 1 +0.562 -0.768 +0.755 +0.894 +0.822 
*:i.:$ *:b* :b** Production 
Vacuolar 1 
(citrate) 
(citrate) 
Citrate 1 
Gradient 
Cytosolic 
PH 
Transtonoplastic 
pH gradient 
-0.369 i-0.778 
:b:b :b:b:i 
-0.678 
:b) 
Cytosolic 1 
**,e 
+0.675 
,w:t  
-0.705 ... -I.*+ 
+0.765 
1 
:wi:t. 
:):w 
+0.640 
:b*:b 
-0.752 
+0.800 
+0.935 
1 
,i** 
:b*:b 
,e:!:! , 
2. Pflanzenphysiol. Bd. 114. S. 269-277. 1984. 
INVERTA 
PEP.carb 
b - ~  
x 
3 
PY R UVAT 
deca rbox 
GAP.deh! 
aY 
L 
6.0 6.! 
l ~ U w . 4  JACOB u
7.0 7.5 8.0 
PH 
Cytosolic homeostasis whitin laticifers 
.i- 50t ;..=* 
271 
Fig. 2: a) Dependence on p H  of invertase, PEP-carboxylase, pyruvate decarboxylase and glyce- 
raldehyde 3-phosphate dehydrogenase from the latex cytosol (activities are expressed in ar- 
bitrary units) as measured in ultrafiltered latex cytosol. - b) Inhibition of latex-cytosolic in- 
vertase by Mg+', phospho-fructo kinase and PEP carboxylase by citrate, and PEP carboxylase 
by Ca", measured in artificial buffers. 
Production of latex reflects the intensity of metabolism within these specialized 
cells. It must be sufficient to regenerate and compensate for the loss of latex upon 
each tapping (generally twice a week). 
Rubber production has been shown to be correlated positively to the pH of the 
cytosol of the latex cell and negatively with the intravacuolar pH (Coupé and Lam- 
bert, 1977; Brzozowska-Hanower et al., 1979). Further, we demonstrated a highly 
significant inverse relationship between the pH of the cytosolic compartment and 
the changes in intravacuolar pH (lutoidic pH), suggesting the existence of some vec- 
2. Pflnzenphysiol. Bd. 114. S. 269-277. 1984. 
272 H. CHRESTIN, X. GDROL, B. 'EUIARIN, J. L. JACOB and J. D'AUZAC 
> 
t 
1 
a 
I- o 
Fig.3: Dependence on p H  of the tonoplastic ATPase and NADH c cytochrome oxidore- 
ductase activities, and of their proton pumping efficiency, measured in buffered ultrafiitered 
cytosol from Hevea latex. 
torial H+ fluxes at the level of the lutoidic tonoplast (Fig. 1) Brzozowska-Hanower 
et al., 1979). 
Multivariate analysis shows that the latex from high yielding rubber-trees is not 
only characterized by a slightly alkaline cytosolic pH and a high transtonoplastic 
H+ gradient, but also by a pronounced accumulation of citrate in the vacuoles, re- 
sulting in a high transtonoplastic citrate gradient, i.e. a low citrate concentration in 
the cytosol (Table 1). 
These relationships could be explained satisfactorily by the extreme pH sensitivity 
(physiological p H  range) of numerous key enzymes of cytosolic metabolism and 
their inhibition by some ions, such as citrate, Ca++, Mg", and CU++, at physiolog- 
ical concentrations (for example see Fig. 2 a-b) (Jacob and d'Auzac 1967, 1969, 1972; 
d'Auzac and Jacob, 1969; Tupy, 1969, 1973; Jacob et al., 1979). 
The transtonoplastic H+ fluxes, the cytosolic as well as the vacuolar pH changes, 
are shown to be under the control of two opposing H+ translocating systems, locat- 
ed at the level of the lutoidic tonoplast (Crétin et al., 1982). 
The first is a tonoplastic ATPase dependent on Mg++ (d'Auzac, 1975 and 1977), 
which catalyses H+ influx into the vacuole causing cytosolic alkalinization and an 
increase in transtonoplastic pH gradient (Marin et al., 1981; Crétin, 1982; Crétin et 
al., 1982; Marin and Blasco, 1982). 
The other is a tonoplastic electron-transporting system, NADH-cytochrome (ar- 
tificial acceptor) oxido-reductase, and may be the same as discovered by Moreau et 
al. (1975). This redox-chain induces H+-efflux, followed by cytosolic acidification, 
and a collapse of the transtonoplastic pH gradient (Crétin, 1983). 
2. P'anzenphysiol. Bd. 114. S. 269-277. 1984. 
Cytosolic homeostasis whitin laticifers 273 
INCREASE 
(%/control) 
Fig. 4: Kinetics of the Ethrel effects on the tonoplastic ATPase potential activity (- . - . - . ), 
the cytosolic ATP level (-) as expressed in YO variations compared with the control, and 
on the vacuolar acidification ( . . . ), in the latex from ethrel treated Hevea bark (base line 
= control). 
Plotting the activity of these two H+ pumps as a function of the p H  of the me- 
dium shows that the ATPase remains at its maximal potential activity over the phy- 
siological range of pH, while the tonoplastic e- transport chain, being p H  sensitive 
(same p H  range), becomes more efficient at a slightly alkaline pH (Fig. 3). This sug- 
gests that an excessive ATPase-induced cytosolic alkalinization will be counteracted 
by the activation of the NADH dependent H+ efflux. 
Moreover, these two H+-pumping systems, which up to now have been charac- 
terized in totally artificial isotonic buffers, have recently been shown to function at 
least as well in <<quasi in vivo, media (fresh latex cytosol deproteinized by ultrafiltra- 
tion on AMICON molecular filter PM 10) (Crétin et al., 1983) suggesting that these 
pumps may indeed function in vivo. 
Treatment of Hevea bark with ethrel, an ethylene generator which <<stimulates, la- 
tex production (d’Auzac and Ribaillier, 1969), induces an increase in the activity of 
the H+-pumping ATPase, probably owing to activation of specific protein synthesis 
at the tonoplast level. The major part of this ATPase activation results in fact from a 
2. P’anzenphysiol. Bd. 114. S. 26%277. 1984. 
274 H. CHRESTIN, X. GIDROL, B. MARIN, J. L. JACOB and J. D'AUZAC 
CPf 
a 
(Ca) 
10 
7 
2 
1 
O 
I 
4 
20 40 60 min. 
Time 
CPm 
.io-3 
(MeA.) 
20 
15 
10 
5 
Fig.5': Effects of addition of (Ca++ + Mg") and the divalent ionophore A-23187, on the 
Ca++ (-) and methylamine fluxes ( .  . 0 )  across the lutoidic tonoplast. Lutoids were loa- 
ded until equilibrium, with 45Calcium or 14C-methylamine (MeA) at pH 7.0; the Ca++ and 
MeA (PH probe) changes were continuously monitored in the external medium by the flow 
dialysis technique (Crétin, 1982). Effectors were added as indicated by the arrows. At the end 
of the experiment lutoids were lysed by triton (0.5 %). 
45 
great increase in the  concentration of the  substrate ATP (Fig. 4), and the appearance 
of some low molecular weight activators in the cytosol (Crétin et al., 1983). This 
activation of ATPase induces a great increase of transtonoplastic A p H  and thus a 
marked cytosolic alkalinization (Tupy, 1973; Coupé et  al., 1976; Coupé, 1977; Brzo- 
zowska-Hanower et  al., 1979) resulting in the activation of metabolism in the  laticif- 
ers. 
Citrate, a potent inhibitor of numerous key  enzymes of the  cytosolic metabolism 
when present in excess concentrations (Jacob and d'Auzac, 1967; Jacob, 1970; Jacob 
et al., 1979) in the  cytosol (a 7mM), is compartmentalized in vivo inside the  latex 
vacuoles (40 to 80 mM in the  lutoids) (Ribaillier et al., 1971; Chréstin, unpublished). 
2. P'nzenphysìol. Bd. 114. S. 269-277. 1984. 
Cytosolic homeostasis whitin laticifers 275 
Fig. 6 The dual role of the lutoidic tonoplast in the control of the cytosolic homeostasis in 
Hevea latex: as a <(biophysical pH-STAT, and a adetoxicating TRAPS compartmentalizing in- 
hibitory ions of the cytosolic metabolism. The figure shows the present state of knowledge 
about the energization of transtonoplastic H+ fluxes and transport of solutes in the lutoids. 
The tonoplast ATPase and <<redox system, function as opposing proton pumps. Data obtain- 
ed from citrate, lysine and Ca" fluxes across the lutoidic tonoplast agree with mechanisms of 
solutes/proton(s) antiporter. The existence of two kinetic pools for accumulated solutes is 
proposed. 
In vivo, the lutoïds accumulate exogenous citrate against a steep concentration gra- 
dient (d'Auzac and Lioret, 1974; Montardy and Lambert, 1977) and any amplifica- 
tion of the transtonoplastic Hi-gradient leads to the transport of citrate into the va- 
cuolar space (Marin, Marin et al., 1981). Mg-ATP is efficient in the energetization of 
citrate accumulation. This ATP-energized influx of citrate is inhibited by proto- 
nophores (Marin, 1981; Marin et al., 1981; Chréstin, unpublished). However at- 
tempts to provoke efflux of the accumulated citrate, by collapsing either or both the 
transtonoplastic p H  and the potential gradients of intact lutoids, in vivo, were inef- 
fective so far (Montardy and Lambert, 1977; Chréstin, unpublished). 
We therefore suggest that the lutoids behave as a mon-reversible detoxifying trap. 
and prevent the inhibition of the cytosolic metabolism by excess of citrate. 
Although Ca2' is necessary for optimal activity of metabolism, if present in the 
cytosol at concentrations > 0.5 mM it inhibits some cytosolic key enzymes (Jacob et 
al., 1979). In vivo, Caii ions are normally compartmented (up to 2.5 mM) inside the 
lutoidic vacuolar space (Ribaillier et al., 1971). 
2. P'nzenphysiol. Bd. 114. S. 269-277. 1984. 
276 H. CHRESTIN, X. GIDROL, B. MARIN, J. L. JACOB and J. D'AUZAC 
In vitro, the  lutoids strongly accumulate Ca++ against a steep concentration gra- 
dient, and Ca++ fluxes always follow the  H+ fluxes i.e. the changes of the transto- 
noplastic pH gradient (Chréstin, unpublished; Marin et al., 1982). Mg-ATP is effi- 
cient in energizing Ca++-accumulation in the  lutoids (Fig. 5) .  Which is inhibited by 
protonophores. Part of the  (free?) Ca++ accumulated is released into the  external me- 
dium if lutoids are treated with divalent cation ionophores (Fig. 5) ,  protonophores, 
acidified medium, or if the  NADH-dependent H+-efflux pump is allowed to work 
(Crétin, unpublished results). 
Consistent with the  functioning of the  two opposing H+-pumps at the tonoplast, 
we propose that the  vacuolar compartment of the latex cells (d'Auzac et al., 1982) 
plays a double role as a <<biophysical pH-STAT, and a adetoxicating trap),, thus con- 
trolling homoeostasis in the cytosol and favouring active metabolism within the 
cells, thus resulting in high latex (rubber) production (Fig. 6). 
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Role of the lutoidic tonoplast in the control of the cytosolic homeostasis within the laticiferous cells of hevea

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