Metabolic regulation and homeostasis in the laticiferous cellhorizon.documentation.ird.fr/exl-doc/pleins_textes/pleins_textes_6/b_fdi_45-46/... · metabolism towards the synthesis

nsferase ice of a further

wferase !ted and guished wght to is sure

SI merit . Brian

t

Fonds Documentaire ORSTOM

Cote : Ex :

165

Chapter 3.HI

METABOLIC REGULATION AND HOMEOSTASIS IN THE LATICIFEROUS CELL

H. Chrestin, B. Marin, J.-L. Jacob, and J. d'Auzac

TABLE OF CONTENTS

I.

II.

III.

IV,

v.

Introduction.. .................................................................. .166

Participation of a Biochemical pH-stat in the Regulation of the pH of Latex Cytosol ................................................................... 166

Participation of a Biophysical pH-stat in the Regulation of Cytosol pH m Latex Cells ................................................................... 168 A. The Inward H' Pumping Function of the Tonoplast ATPase.. ......... .169 B. The Outward H' Translocating Activity of Tonoplast NADH-c-

Cytochrome Reductase ................................................. .170 C. Further Evidence for Location of Both H+-Pumps on Lutoids ......... .171 D. The Tonoplast pH-stat: Regulation of and by pH.. ..................... .171 E. Interaction Between Biochemical and Biophysical pH-stat on

the Regulation of Cytosol pH and Metabolism ......................... .172

Control of the Ion Composition of the Cytosol by Lutoids.. ................... .172 A.

B.

Energization of Citrate Transport and Accumulation at Lutoid

Energization of Calcium Fluxes at Lutoid Tonoplast ................... .176 Tonoplast.. ............................................................. .173

Conclusion.. ................................................................... ,176

c

166 Physiology of Rubber Tree Latex

boxyla pender

I . INTRODUCTION

When there is no major difficulty in latex flow (see Sections 4 and 6) it can be considered that production of rubber reflects the intensity of the metabolism within these specialized cells that constitute the laticiferous system. Obviously, at least as far as exploited Hevea are concerned, the regenerative metabolism within the laticifers must be sufficiently active to compensate for the loss of cellular cytoplasm (i.e., latex) between each tapping.

The optimal regenerative metabolism, and above all the preferential orientation of the metabolism towards the synthesis of natural rubber in the laticiferous cells, is governed by numerous factors. In particular, the regulation of pH and ionic composition of latex cytosol in which glucidic catabolism and almost all isoprenic synthesis take place, have been shown to be of prime importance (Section 3).

This was shown in particular by the demonstration of a highly significant direct relationship between rubber production and the pH of latex ~ y t o ~ o 1 . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . ~ ~ ~ The greatest amplitude of cytosol pH variations (seasonal and tree by tree) was shown to be 0.4 pH units above and below a mean pH value of about 6.9. It was then obvious that there were complications in the regulation of the laticiferous metabolism by the pH and probably also in the regulation of the pH by the intracellular metabolism.

This high dependency of rubber production on cytosol pH was explained satisfactorily insofar as numerous key enzymes involved in glucidic catabolism were shown to be highly pH-sensitive (Chapter 3.1). Indeed, it was clearly established that slight variations (alkalinization) in cytosol pH in the physiological range induced considerable activation of glucidic catabolism and then enhanced rubber production through the latex invertase activity which controls the s t q of sucrose catabolism and then latex regeneration and whose optimum pH displays a shai-p peak.467.470

Other key enzymes were also shown to exhibit high pH dependency. This was the case of pyru~ate-decarboxylase~~~~~’ which converts pyruvate into acetate, the obligatory pre- cursor of rubber synthesis, and of glyceraldehyde-3-phosphate dehydrogenase (Figure 1A). Likewise, it was demonstrated that phosphoenolpyruvate carboxylase (PEPcase), which can divert glycolysis from the “isoprenoid pathway” to the synthesis of certain Krebs cycle acids, is particularly sensitive to physiological pH changes (Figure 1A).259*4E1 It was then concluded that the regulation of cytosol pH was of capital importance for biogenesis of rubber in latex cells.

Solute absorption and metabolism in plants leads to the production or consumption of H+ or OH-. Fortunately, it is generally accepted that there may be at least two types of process able to remove solutes (strong acids or bases, H+ or OH-, etc.) which are able to modify the pH of a cellular compartment beyond physiological limits. One type is a chemical or biochemical process which involves the conversion of one or more solutes into a chemically different form in the same cellular compartment (biochemical pH-stat). A second process, known as a “biophysical” or “bioosmotic pH-stat” consists of the removal of these solutes by their translocation through the membrane that separates two subcellular

It is accepted that these two types of process are not mutually exclusive, but .on the contrary may occur simultaneously or successively throughout the metabolic pathways. They may depend on one another or at least mutually influence each other and may in some cases be coupled by an energy-dependent process according to the chemiosmotic theory proposed by M i t ~ h e l I . ~ ~ ~ . ’ ~

II. PARTICIPATION OF A BIOCHEMICAL PH-STAT IN THE REGULATION OF THE pH OF LATEX CYTOSOL

The central role of the “phosphoenolpyruvic acid crossroads” in the regulation of pH and plant metabolism was pointed out by da vie^.'^^'^* Davies’ theory is based on a car-

neutral precur! (glucid

‘.,

Stud. and py- mecha!

I

B z - i .

d

INVERTACE

,i ?

I if PEP.carboxyi8se

I #' i m

r l i - l

7,

RRUVAT; \+

\

\

jecarboxylask,

G AP.deh/drogenaso

I I I A

,P' a'

I I

I

,A' .if

-4'

167

/p, 0.

I I

FINa /P

I

3

, I

I 1

,A*

B iidered ialized Hevea active

of the ned by

shown

onship olitude above

cations dation

~ytosol

ctorily highly (alkal- iucidic which im pH

e case Y Pre- c: IA). ch can cycle s then

s i s of

of H' mess iodify cal or iically xess , slutes its. 425

j n the =Y cases m e d

ION

>f pH 1 car-

\ A I /..........*"

INVERTASE a*C...-- 8.5

1 5 10 2 0

~.

I 1 I I O 50 6.5 7.0 7.5 8.0

PH

-A- PEP-carboxylase *_----

I *

,w' ,.**

,,A'

J I I k a * ) m M I

0.5 1 2

RGURE I . The imporiance of the regulation of cytoplasmic pH (A) and of the concentration of some cytosolic ions (B) on the activity of some cytocolic key enzymes. The vertical lines of Figure IA illustrate the physiological variations of cytosolic latex pH.

boxylation-decarboxylation system controlled by enzyme activities with very high pH-dependency.

(acid pH) I I (neutral or alkaline pH)

neutral PEPcase I (maiic enzyme) precursors - PEP -7% OAA 1. Malate 7% Pymvate (glucids) CO, + OH- I CO2 + OH-

I

Schematic representation of the phosphoenolpynivic crossroads as involved in metabolic pH regulation in plant cells, according to Davies.'M

Study of the enzymes which constitute the phosphoenolpynivic crossroads (PEPcase2@ and pynivate kinase263 in latex) made it possible to demonstrate the existence of regulation mechanisms which play a role in controlling cytosol pH.


As measured in latex cytosol, PEPcase leads via oxaloacetate (OAA) to the rapid formation of malate and citrate.240 Furthermore, it was shown that both malate and to a lesser extent citrate act as effective inhibitors of PEPcase but that this inhibition is highly pH-dependent.259.m When present at a sufficiently high concentration, malate inhibits PEPcase to a greater extent at a slightly acid pH than at a slightly alkaline one (Chapter 3.1). This results in a pH-sensitivity of the carboxylase which depends on the concentration of malate. Thus, with a sufficient concentration of malate in the cytosol, slight alkalinization will induce strong activity and, on the contrary, slight acidification will result in effective inhibition of carboxylase. From this it was concluded that the malate content (> 7 mM) of the latex cytosol and the physiological variations of the cytosol pH would effectively modulate the carboxylation of PEP.

Insofar as it has been demonstrated with certainty that the functioning of PEPcase in latex cytosol does produce organic acids (malate and citrate), accumulation of the latter can induce significant acidification of the cytosol, resulting in self-regulation which tends to inhibit enzyme activity itself. Thus in relatively acid cytosol (pH 6.6 to 6.8) it was shown that the production of OAA, malate, and citrate from PEP and I4CO, is extremely small. On the contrary, from pH 6.8 onwards carboxylation of PEP in latex cytosol was shown to lead to the synthesis of relatively large amounts of malate and citrate tending to limit further alkalinkation of the c y t o s ~ l . ~ ~ . ~ ~ ~ Furthermore, metabolism or removal of these acids from the cytosol by translocation to another subcellular compartment (the vacuole) will initiate the process once again. The very low buffer capacity of latex cytos01"~ is worth noting since slight variations of concentrations of H' and OH- will bring about important changes in cytosol pH. The first part of the pH-stat mechanism mentioned by Davieslm and then by Smith and

Raven= thus appeared to be physiologically functional in latex cells. The second part of the mechanism implies the irreversible decarboxylation of malate by malic enzyme into a weaker acid (pyruvate) and CO,, which results in a realkalinization of the medium. The maiic enzyme has been shown to be present in latex cytos01,~~~ and to be functional within the limits of availability of NADP'. It was therefore concluded that ail the elements able to contrïïute to the functioning of a Davies biochemical pH-stat do exist and might be functional in physiological conditions.

Besidts this biochemical the pH-stat, processes removing H' by translocation through membranes bounding the cytosol compartment (including plasmalemma and tonoplast) constitute a mechanism of major importance which contributes to the regulation of cytosol pH in plant cells. The most important processes leading to transplasmalemmic fluxes of protons or other ions in the plant kingdom have been reviewed As far as the higher piants are concerned, the action of plasmalemmic ATPase in the processes controlling extniskm of H+ was demonstrated recently. 123,3'57447.491

Unfatunately, because of the purely cytoplasmic nature of latex (i.e., it consists of cell contents), plasmalemma is not carried out with latex flow and thus could not be obtained and purified in such investigations.

III. PARTICIPATION OF A BIOPHYSICAL pH-STAT IN THE REGULATION OF CYTOSOL pH IN LATEX CELLS

Furtha investigation of the physiological parameters involved in latex production showed that niWer production was not only linked to the pH of latex cytosol (Figure 2A) but also associattd positively with the transtonoplastic pH gradient (the difference in pH across the membrane separating the cytosol from the intravacuolar medium) and negatively with the intravacn~lar PH.%*^^'*'^

Monover, a highly significant inverse relationship was demonstrated, linking the pH of the cytasolic compartment and the intravacuolar pH (lutoidic pH) (Figure 2B), suggesting

I

.- I

r C

I; c

I

i

t

I

l

t r i

such genen inizati This 1i and th 'and th tonop betwet

A. Th The

in rho:

of citr Dei

obtair

mediu. lipoph isolatt. electrc pheno inan transn

1 an inc

b' d'A

of Mp

Probe

* Ab}

iation 2xtent pend- . to a esults Thus, 7duce Ion of latex

'e the

i latex nduce nhibit at the

ln the ead to urther from

Titiate ioting anges

h and art of into a

I . The Mithin s able ht be

pH.L 6.5

6.0

5.5

5.0

lrough ) con-

-otons 3s the olling

-01 pH

-

-

-

-

-

-

I I I I I

)f cell ained

rION

owed also

ss the th the

pH of =sting

l

i

í 1

I

, i . . ' 1

I

. ' i , ,

I ! i

i

i ~

i

i I i

l

i I

I ! I I

j !

1

\ l i

-- c I m

'5 50 - O 4-

c

1 40 e d, .. 30 a w

- .I-

- -I

>. 20 -

10 -

I %

169

6.4 6.6 6.8 7.0 1 2 6.0 6.5 7.0 pH.C C SERUM pH .

FIGURE 2. (A) Relations between the pH of the cytoplasmic C serum and the yield and (B) between the pH of cytoplasmic serum and the pH of lutoidic serum (pH L). (Adapted from Brzo- zowska Hanower, J., Cretin, H., Hanower, P., and Lioret, C., Physiol. Vég., 17, 889, 1979.)

the occurrence of vectorial H + fluxes at lutoid membrane (tonoplast) level. In support of such a hypothesis it was shown that treatment of Hevea bark with Ethrel@, an ethylene generator which "stimulates" latex production (see Chapter 5.11), induced distinct alkalinization of the latex ~ y t o ~ o 1 ~ ~ * ~ ~ ~ . ~ ~ ~ accompanied by marked acidification of the vacuole.%*14' This led to seeking mechanisms which might be able to control transtonoplastic proton fluxes and then regulate cytosol pH and metabolism. The ATPase activity showed by ~ ' A u z ~ c ~ ~ . ~ ~ and the redox system discovered by Moreau et al. ,347 both of which are located on the lutoid tonoplast, were suspected of being good candidates for controlling such proton exchanges between the cytosol and vacuolar compartment in latex cells.

A. The Inward H+ Pumping Function of the Tonoplast ATPase The role of a lutoid ATPase as a possible H' pump had been foreseen as early as 1974

by d'Auzac and LioreP and found positive arguments in the results of Lambertz8' and then in those of Hanower et al.zo8 showing significant acidification of the lutoids upon the addition of Mg-ATP to a suspension of isolated lutoids in order to stimulate vacuolar accumulation of citrate or basic amino acids in vitro.

Definite proof of the proton-pumping function of the lutoid tonoplast ATPase was first obtained by Marin et al.323,33z using I4C-methylamine as a transmembrane proton gradient probe withe tonoplastic vesicles obtained from freeze-dried tonoplast in a totally artificial medium. Using the same ApH probe and either 9ubidium (with valinomycine) or the lipophilic cation tetraphenylphosphonium as a transmembrane potential probe with fresh isolated native lutoids, Chre~tin"'*'~' showed that tonoplast ATPase activity caused an electrogenic (uncompensated charge) influx of protons in lutoids. Quantification of these phenomena (Table 1) showed that addition of Mg-ATP (3 mM) to fresh lutoids in suspension in an artificial medium at pH 7.0 induced vacuolar acidification of about 0.55 pH unit and transmembrane depolarization of about 48 mV (A+: interior less negative), thus leading to an increase in the transtonoplast electrochemical proton gradient A&H+ (see footnote*) of

* A&H+ = A$-Z ApH, in which Z = 2,3 RTE = 59 at 25°C when expressed in mV.


Table 1 COMPARATIVE STUDY OF THE

TRANSTONOPLASTIC ApH, ApH+, A*, AND OF INTAlCT LUTOIDS LOADED IN ARTIFICIAL BUFFER

ORULTRAFILTEREDLATEXCYTOSOLINTHE PRESENCE OF ABSENCE OF Mg-ATP

- Z.ApH (mV) A Y (mV) A&H+ (mV)

Medium None +ATP None +ATP None +ATP

Buffer 70 101 -68 -20 + 2 + 81 Cytosol 82 145 -28 - 2 +54 + 143 Cytosol + FCCP 40 33 -87 -97 -47 - 59

Adapted from Chrestin, H., Le compartiment vacuo-lysosomal (les lutoides) du latex d'Hevea brasilienris. son rôle dans le maintien de I'homeostasie et dans les processus de senescence des cellules IaticiRres, Thèse Doct. Etat Sci. Nat., Université Montpellier II, 1984, et Collection (Etudes et Thèse), O.R.S.T.O.M., Ed., Paris, 1985.

about 80 mV. The addition of a protonophore such as FCCP reduces the ApH and the A&H+ while inducing hyperpolarization.

teinized latex cytosol (obtained by ultrafiltration on PM 10 Amicon membrane) that the addition of Mg-ATP effectively resulted in acidification of the vacuole and transmembrane depolarization followed by an increase in the transtonoplast A&H+ of the same magnitude as in a totally artificial medium (Table 1).115.118*186

It was then concluded that the ATPase located on the lutoid tonoplast could really be functional in vivo. Finally, since the K, of lutoid ATPase and its H+-pumping activity were shown to be about 0.5 mM under physiological conditions (ultrafiltered latex cytosol), lE6

and since the mean ATP content in the cytosol remained less than 0.2 mM in latex from nonstimulated trees (see Chapter 5.11), it was suspected that in vivo the tonoplastic H+- pumping ATPase always operates at far less than its maximum potential and remains con- stantly controlled by the availability of ATP in the latex cells.115JL8 Nevertheless, with a constant supply of ATP provided by the whole metabolism in latex cells, the proton pumping ATPase must be effective in maintaining a high transtonoplast proton gradient and in controlling the pH of the cytosol in a manner that consumes energy.

Moreover, it was demonstrated with freshly purified native lutoids resuspended in depro- .

B. The Outward H+ Translocating Activity of Tonoplast NADH-c-Cytochrome Re- ductase

More recently, an outward H+-pumping function of a redox chain located on the lutoid tonoplast was discowered; this is probably the same as the one reported by Moreau et Indeed, it was that addition of NADH to a suspension of freshly isolated intact lutoids in the presence of an artificial acceptor such as cytochrome c or potassium ferricyanide resulted in an efflux of protons from the lutoids followed by vacuolar alkalinization and, conversely, acidification of the external medium and then considerable reduction of the transtonoplast proton gradient. Transmembrane potential probes were used to that the functioning of this tonoplast redox chain resulted in transmembrane hyperpolarization (inside more negative) and then reduction of the transtonoplast electrochemical gradient of H+ . These results are also confi ied by utilization of FCCP.

The protonbumping activity of the redox chain was also shown to be functional in vivo (lutoids suspended in ultrafiltered cytosol) (Table 2), at least when the medium was com- plemented with exogeneous electron acceptors.

I

depen translc displa Neithi braneb with c malatc in fres and th conclu

D. Th ~

Whc of the ATPa (6.6 ti Pump' efficii excess H + -p depenl retical

A&H+

depro- .hat the mbrane jnitude

ally be ty were )SOI), '86

x from ic H+- ns con- with a

umping in con-

ne Re-

* lutoid :t al. 347

1 'intact :yanide m and, of the

'46 that ization

Aient of

in vivo .s com-

171

Table 2 COMPARATIVE STUDY OF THE TRANSTONOPLASTIC ApH AND

ApH+ OF INTACT FRESH LUTOIDS LOADED IN ARTIFICIAL BUFFER OR ULTRAFILTERED LATEX CYTOSOL, IN THE PRESENCE OR ABSENCE OF NADH + CYTOCHROME C

- Z.ApH (mV) AVr (mV) AfiH+(mV)

+ NADH + NADH + NADH Medium None + cyt. c None t cyt. c None + cyt. c

Buffer 68 . 47 - 80 -102 - 12 - 55 Cytosol 73 42 - 37 - 63 t-31 -II Cytosol t FCCP 41 36 -100 - 93 - 59 - 57

Adapted from Chrestin, H., Le compartiment vacuo-lysosomal (les lutoides) du latex d'Hevea brusiliensis, son rôle dans le maintien de I'homeostasie et dans les processus de senescence des ecllules laticieres, Thtse Doct. Etat Sci. Nat., Universiti Montpellier II, 1984, et Collection (Etudes et Thtse), O.R.S.T.O.M., Ed., Paris, 1985.

Partial characterization of this proton-pumping redox chain showed that it was not affected by the classic inhibitors of cytochrome respiratory chains (KCN, antimycin A, etc.) or by those of the mitochond,rial alternate pathway (hydroxamic acids, propyl galate)."5J46 It was then concluded that this redox chain activity could not be attributed to any mitochondrial or bacterial contaminant.

C. Further Evidence for Location of Both H+-Pumps on Lutoids Whereas solid evidence was reported showing that the two opposing H + -pumps were not

of bacterial, mitochondrial, or plasmalemmic origin,' 15.186~188*323 it might have been thought that one or other proton-pumping system might be located on different sedimentable organelles in latex (Frey-Wyssling particles, microsome-like entities, vesiculated endoplasmic

showed that ATPase activity and the ATP- dependent H+-pump, together with the NADH cytochrome c oxidoreductase and the H+ translocating system associated with the latter, were superposable on the density fraction displaying molybdate inhibitable acid phosphatase activities, a typical lutoid marker.372.390 Neither of the proton pumps, which necessarily characterize only intact organelles (membranes must be completely intact for transmembrane fluxes to be detected) could be associated with o-diphenol-oxidase (a marker of Frey-Wyssling particles) or with the sedimentable malate-dehydrogenase (a marker of as yet unidentified medium-dense membrane particles in fresh Furthermore, it was confirmed that both enzymatic activities (i.e., ATPase and the redox system) were located on membrane It was thus definitely concluded that both opposing proton pumps were located on lutoid tonoplast.

\ reticulum, etc.). Isopycnic

D; The Tonoplast pH-stat: Regulation of and by pH When the activity of these two opposing H+-pumps was plotted as a function of the pH

of the medium (buffered ultrafiltered cytosol), it was shown that the inward H+-pumping ATPase remained at almost maximum potential activity over the physiological pH range (6.6 to 7.2), while the tonoplast electron transport chain and its associated outward H+- pumping activity, which is much more sensitive over the same pH range, became more efficient at a slightly alkaline pH (over pH 7.3)."5.'20*'* It was then suggested that any excessive alkalinization of latex cell cytosol, possibly through excessive functioning of the H+-pumping ATPase would be counteracted effectively by the activation of the redox- dependent efflux of H+ (Figure 3). This would mean that pH of the cytosol would theo- retically tend to stabilize itself in the pH range lying between the optimum pH of each proton


.

, FIGURE 3. Dependence on pH of the lutoidic ATPase and NADH-c cytochrome- oxidoreductase activity and of their proton pump efficiency measured in buffered ultra- filtrated cytosol from Heveu latex. (Adapted from Chrestin, H., Gidrol., X., Marin, B., Jacob, J. L., and d'Auzac, J.. Z. Pflonzenphysiol., 114, 269. 1984.)

pump, providing that the availability of their respective'substrates was not a limiting factor in latex. This system is a typical biophysical (bioosmotic) pH-stat based on controlled energy- consuming transmembrane fluxes of free protons. IIs

E. Interaction Between Biochemical and Biophysical pH-stat on the Regulation' of Cytosol pH and Metabolism

There is little doubt that because of the strong specific activity of PEPcase and the very weak buffering capacity of latex cytosol and because of the existence of malic enzyme within latex, the altemative phosphoenol-pyruvate pathway plays an important role as biochemical pH-stat in latex cytosol.

The key role of malate, the major strong acid product of the PEPcase pathway through NADH-dependent malate-dehydrogenase should be noted. It was shown that malate ensured permanent sensitization of PEPcase to pH, so that any acidification of the cytosol (possibly caused by accumulation of malate through the PEPcase pathway) in the presence of this strong diacid would bring about effective inhibition of the PEPcase itself. Furthermore, malate has been shown to act as an effective activator of lutoid tonoplast ATPase at physiological concentration^.^^*^^*'^^ It is therefore suggested that any slight acidification of the cytosol (in the physiological pH range corresponding precisely to that of the optimal pH of the H+-pumping ATPase) in the presence of, or caused by malate will bring about further activation of the proton-pumping ATPase. This would, on the one hand, re-alkalinize the cytosol and thus activate the primary metabolism, and on the other hand, through ATPase activity cause the accumulation of ADP - a prime regulator of pyruvate-kinase, hence favoring the relative activities of PEPcase and pyruvate-kinase in the direction of the pyruvate pathway.

IV. CONTROL OF THE ION COMPOSITION OF THE CYTOSOL BY LUTOIDS

T

Ck Cl

Late Pro

Vaci (cit

cyto (cit

citra g"

Cyto PH

PH Tran

Not:

(***

(**).

2.11). as a 1

Pi, M effec case, Th

metal set al from and i the v gradi show

A the m: that tl

of citr' fully (

A. En As

descri Liorei on ton vesiclt The kl linear

Like all plant vacuoles, lutoids accumulate numerous ions in addition to H+ (see Chapter

I

. *a

factor nergy-

tion of

!e very within emical

hough mured xsibly of this rmore, i phys- of the

. pH of further ize the LTPase hence

Tuvate

Jhapter

173

Table 3 TABLE OF THE CORRELATION COEFFICIENTS LINKING THE LATEX

PRODUCTION (GRAMS DRY RUBBERPTAPPINGPTREE), THE LATEX CYTOSOLIC pH, THE TRANSTONOPLASTIC pH GRADIENT, THE LATEX CYTOSOLIC AND VACUOLAR CITRATE CONCENTRATIONS ("), AND

THE RESULTING TRANSTONOPLASTIC CITRATE GRADIENT"S~'zO

Latex Vacuolar Cytosolic Citrate Cytosolic Transtonoplastic production (citrate) (citrate) gradient pH pH gradient

Latex production

Vacuolar (citrate)

Cytosolic (citrate)

Citrate gradient

Cytosolic PH

Transtonoplastic pH gradient

1 + 0.562 - 0.768 + 0.755 + 0.894 - 1 - 0.369 + 0.778 + 0.675 - - 1 - 0.678 - 0.705

*** *** *** ***

** *** ***

*** *** - I + 0.765 - -

***

+ 0.822 + 0.640 - 0.752

***

***

*** t 0.800

+ 0.935

1

***

***

Nore: Data obtained from freshly collected latex of 56 rubber trees.

(***), Very high significance. p SO.01. (**). High significance. p S0.05.

2.11). Compartmentation in vacuoles of certain effectors of cytosolic enzymes can be regarded as a mechanism which participates in the regulation of the cytosol metabolism. Indeed, the Pi, Mg, Ca, Cu, citrate, and malate that accumulate in lutoids were identified as physiological effectors of various cytosol enzymes (Figure 1B) such as NADP-ph~sphatase,"~.~~~ PEP- case,26s p~ruvate-kinase,~~~ invertase,zs and malic enzyme.253

The influence of metabolites and ion compartmentation on the intensity of the cytosol metabolism were revealed in detail by studies of the compartmentation of solutes in latex set against production of rubber. In particular, multivariate analysisLts showed that latex from high-yielding rubber trees was not only characterized by a slightly alkaline cytosol pH and a high transtonoplast proton gradient, but also by a marked accumulation of citrate in the vacuoles and a low citrate content in the cytosol resulting in a high transtonoplast citrate gradient (Table 3). This was satisfactorily explained since certain key cytosol enzymes were shown to be inhibited by physiological concentrations of citrate (Figure 1B).

A feature of these data was the very highly significant direct relationship linking in vivo the magnitudes of the transtonoplast citrate and proton gradients. It should be pointed out that this relationship between transtonoplast ApH and citrate gradient established in vivo fully corroborated the results of studies of the energization of transport and accumulation of citrate at the lutoid tonoplast as studied in vitro.LL5*323.327-331

A. Energization of Citrate Transport and Accumulation at Lutoid Tonoplast As it had been clearly shown that citrate accumulates in lutoids in vivo,3w attempts to

describe the transport of citrate at lutoid tonoplast in vitro were made first by d'Auzac and Liorets2 and then by Montardy and Lambert3& on intact native lutoids, and then by Marin323 on tonoplast vesicles reconstituted from freeze-dried lutoids. Both native lutoids and tonoplast vesicles were shown to accumulate exogenous citrate against a steep concentration gradient. The kinetic parameters were clearly defined: citrate uptake was temperature-dependent and linear for at least 30 min, even in the absence of any metabolic energy supply. Its initial


.. 160 t

Oh I I I I

- 150 -100 -50 O + 50 .k 1I mV

.. FIGURE 4. Relations between the components of the proton motive force and citrate incorporation by vesicles obtained from lutoid membrane. Proton motive force (PMF), Ap = APH+ = A'P- y ApH. (Adapted from Marin, B., Cretin, H., d'Auzac, J.. .Physiol. Veg., 20, 333, 1982.) 1

rate in function of citrate concentration in the medium was shown to display simple Michaelis- Menten kinetics with an apparent K, of 7 mM. It was strictly pH-dependent with an optimal value of about 7.0 (i.e., in the physiological pH range) where the thrice dissociated form predominates. 323

The addition of Mg-ATP to the incubation medium was shown to cause a Considerable increase in the magnitude of citrate uptake. The same results were obtained when the lutoids were preincubated with this compound.287 The steady state level of citrate uptake and accumulation in the presence of Mg-ATP was shown to be two to five times higher than the level obtained in the absence of any source of energy. Furthermore, the addition of chemicals able to dissipate the transmembrane pH gradient (protonophores) caused a considerable reduction in citrate uptake in the absence of energy supply and completely halted activation by Mg-ATP. Finally, it was shown that all the known inhibitors of the lutoid tonoplast ATPase inhibited activation of citrate uptake in the presence of Mg-ATP.324.325*331*332

It was then concluded that the energy required for citrate uptake by lutoids, which operates against a steep concentration gradient, originated in the transtonoplast proton gradient and resulted from the functioning of the H+-pumping ATPase located on the lutoid tonoplast. Moveover, it was shown that any change in magnitude of the transtonoplast proton gradient or in the transtonoplast ApH induced parallel changes in the magnitude of citrate uptake (Figure 4), and it was definitely concluded that both components of the proton-motive force, and then A&H+ itself, were involved in the energization of citrate uptake.

Many attempts were then made to characterize any mechanism able to control efflux of the citrate accumulated in the lutoids in order to find out what role it played in the latter. Whatever the technique u ~ e d , " ~ ? ~ and whatever the strategy adopted to try to induce efflux of citrate from intact, freshly isolated lutoids, such as changes in the transtonoplast H+ gradient by external pH variations, use of protonophores, functioning of the outward H+- pumping tonoplast redox chain, changes in transtonoplast potential using KCl + valinomycin or lipophilic cations (MTPP+ , TPP+) in the presence or absence of various concentrations of exogenous citrate in the medium and at three different temperatures (20,30, 40°C), neither of the authors was able to find evidence of any significant efflux of citrate accumulated in vivo or in vitro by intact l~toids ."~ It was then concluded that almost all the citrate which

had ac thus a

In c evider This t citrate the is( activil

Fro that tl kinetil the vi was shc and in in the ( efflux I (i.e., P factors in vitrc

Ther, tonoplh regener

haelis- )ptimal .l form

lerable lutoids ind ac- ian the .micals Jerable ivation noplast

perates mt and .oplast. radient uptake

6 force,

’flux of . latter. efflux

1st H+

omycin trations neither ated in : which

32

rd H+-

175

fil!‘TE\ INHIBITORS

ADP 4

NAD +

oxydo -reductase

FIGURE 5. Schematic representation of the role of tonoplast and lutoids in the cytosol homeostasis of Hevea latex. Lutoids play a double role as a biophysical pH- stat and as a detoxicating trap for compartimentalization of inhibitory ions of the cytosolic enzymes. The tonoplast ATPase and NADH-redox system function as opposing proton pumps. The first of these two pumps generates a proton motive force able to energize soluteslprotons antiporter. The existence of two kinetics pools for accumulated solutes is proposed.

had accumulated remained definitively entrapped in the native lutoids, and that there was thus a true detoxification process (Figure 5).

In contrast, Marin,323.325 working with tonoplast vesicles from freeze-dried lutoids, found evidence of a massive efflux of citrate: up to 80% of the citrate which had accumulated. This efflux was shown to be temperature-dependent and to increase with the amount of citrate in the external medium. However, when labeled citrate was taken up by these vesicles the isotopic enrichment of the internal compartment did not exceed 8% of extemal specific activity and, as a result, isotopic equilibrium was never attained.

From these data obtained on native vacuoles and tonoplast vesicles, it was finally concluded that there might be intemal “compartmentation” of the vacuolar citrate into two distinct kinetic pools. A minor pool was directly interchangeable and the major one (over 90% of the vacuolar citrate) was assumed to remain sequestered in the native lutoids. Insofar as it was shown that there are quasi-stoichiometric concentrations of citrate and Mg+ + in latex, and in particular in luto id^,"^*^^^ it was proposed that vacuolar citrate might be sequestrated in the complex form citrate2- Mg2+ in intact l ~ t o i d s . ~ ~ ~ . ~ ~ ’ It was suggested that the massive efflux of citrate from tonoplast vesicles observed by Marin was to a certain extent an artifact (i.e., nonphysiological) and might have been caused by the loss of intralutoid sequestrating factors, including Mg2 + , during tonoplast vesiculation from freeze-dried lutoid membranes in vitro.

There was no doubt that the dynamic nature of citrate exchanges through the lutoid tonoplast was an important feature of the regulation of the metabolism and then in latex regeneration and hence rubber p rod~c t ion .~~ These transtonoplast citrate fluxes were finally


shown to be closely controlled by the transtonoplast electrochemical proton gradient gen- erated by the differential functioning of the two opposing electrogenic H+-pumps located on the tonoplast itself. Such a situation might account for the links between rubber production and the transtonoplastic H+ and citrate gradient in latex.'15.1j0

B. Energization of Calcium Fluxes a t Lutoid Tonoplast Divalent cations also accumulate in lutoids in vivo.39o They are effectors of numerous

enzymes in the latex cytosol and can either act as activators (e.g., Mg2+ for ATPase and eno1ase,42*240 or Ca2+ is a classic activator of phosphorylating enzymes) or as inhibitors of key enzymes (Figure 1B; Mg2+ for invertase2"0 and Ca2+ for PEPcase268). The energization of Ca2+ fluxes across the lutoid membrane was studied by Chrestin as a model."' It was then shown that Ca2+ is absorbed to a great extent by intact native lutoids in vitro even in the absence of any source of metabolic energy. The rate of Ca2+ uptake was linear, at least during a 45 min period, and was thermodependent. This phenomenon could not have been caused by a mere isotopic exchange or by major cation adsorption on external tonoplast sites. It was shown by various experiments that Ca2+ transtonoplast fluxes were at least partially reversible and were related to variations in transtonoplast ApH. I 15.120 Moreover, solid evidence was obtained which suggested that Ca2+ accumulates in the vacuoles at the expense of the transtonoplast pH gradient. 'I'

Furthermore, the author showed that the working of the tonoplast H+ pumping ATPase induced additional accumulation of Caz + in the lutoids. ATP-energized accumuration was inhibited or reversed in the presence of protonophores,"'*33' or ionophores specific to divalent cations (A-23187). However, only a small proportion of the Ca2+ accumulated in the absence of energy supply can be released into the incubation medium by these ionophores which are specific to the divalent cations.'16 Furthermore, it was shown that a large proportion of the intralutoid Ca2+ could be released into the medium in the presence of membrane depolarizing agents (TPP' , K+ + valinomycin) which contributed to the discharge of the transtonoplast Donnan potential. 145.331*335 Finally, it was shown that the operation of the tonoplast electron transport system, which induced transmembrane efflux of protons, led to the release of a large proportion of the intralutoid Ca2+. The latter phenomenon was shown to be more marked when accumulation of Ca2+ had previously been energized by a supply of ATP."'

All these results led to supposing that there were two Ca2+ pools in lutoids (Figure 5): a major pool entrapped by adsorption on intravacuolar structures and a pool of free, mobile Ca2 + accumulated at a thermodynamic imbalance within lutoids (dissipated by protonophores and divalent cation ionophores). Chrestin"' proposed the existence of a transport system involving a Ca2+/H+ antiport on the tonoplast, and considered that the transtonoplast fluxes of free calcium were controlled by the two opposing H+ pumps located on the lutoid tonoplast.

Finally, it was concluded that the lutoid tonoplast was able to contribute actively to the regulation of the free Ca2+ content of the cytosol to 0.1-1 mM for the regulation of the enzymes mentioned above or more probably, to a very low level (>I. M) compatible with its possible role as a second hormonal messenger, as is recognized t ~ d a y . * ~ . ~ ~ '

V. CONCLUSION

Control of the internal metabolism of latex cells by intracellular pH and the ionic composition of the cytosol has attracted growing interest in recent years because of the predom- inant impact on rubber production (for a review see Jacob et al.273).

It is considered classically that two process& have been developed by plant cells to counteract these ineluctable changes in cytosol pH caused by intracellular metabolism. One is a biochemical pH-stat based on the intracellular production or consumption of H+ . Such

a bit dem carb amo cont

, the] bioc late:

Ir mali the that latic

II con the new undc OPPC this Furt cou; prot the I

M up h

lient gen- 's located îoduc tion

iumerous .'Pase and ibitors of .rgization l5 It was

o even in r, at least lave been tonoplast e at least loreover, les at the

I ATPase dion was i divalent 3 absence ylhich are 3n of the olarizing tonoplast L electron :ase of a be more . ATP. I l 5

:Ure 5): a , mobile nophores t system st fluxes

.le lutoid

ly to the m of the mpatible 335

nic com- ' predom-

cells to sm. One -I+. Such

177 ---- L A T E X VESSEL ----- _ _ _ _ _ LATEX CYTOSOL _ _ - _ _ _ ATP ,. sucrose

n +

NADH LUTOID DAA e PEP

malic oc 3 pyruvote

I NAD+

H+ & cis - polytsoprene

FIGURE 6, Schematic representation of a laticiferous vessel and of some plasmalemmic and tonoplastic enzymes and carrier; some cytoplasmic enzymes implicated into latex homeostasis. ( I ) Latex cell wall; (2) plasmalemma; (3) metabolic pathway leading to cis-polyisoprene biosynthesis; (4) Davies type pH-Stat; (5) plasmalemmic H+-pump ATPase; (6) tonoplast; (7) tonoplastic H+ -pump ATPase (influx); (8) tonoplastic NADH- oxydoreductase H+ pump (efflux); (9) NADH-O, reductase (NADH-Qui- none reductase); (IO) possible linking between 9 and IO; ( I ) H+-solutes antiporter on the tonoplast.

a biochemical pH-stat,'50.'52 centered on the phosphoenol-pyruvate crossroads, has been demonstrated in latex. There is therefore no doubt that the highly pH-dependent latex PEP carboxylase is involved in the regulation of cytosol pH. However, there remains a certain amount of controversy about the true effectiveness of such a Davies pH-stat'in the fine control of cytosol pH because of the relatively low PEPcase:PK activity ratio in latex, and the parallel (instead of opposite) changes under quasi-in vivo conditions. 266 However, these biochemical pathways are probably able to participate in regulation of the pH balance in

In addition it is probable that intracellular pH is governed through control of transplas- malemma H+ transport, although it was not possible to obtain experimental data owing to the unsuitability of mature Hevea material for such studies. Nevertheless, it is very likely that a H+-pump ATPase and some symports for sucrose, K + , etc. are located on the laticiferous cell plasmalemma (Figure 6) .285

It should be pointed out that a new concept of the regulation of intracellular pH and ionic composition in plant cells is beginning to emerge from the results obtained from studies of the interactions between vacuolar; and cytosolic compartments in latex cell cytoplasm. This new concept is based on the active regulation of transtonoplastic H+ fluxes which remain under the control of two H' translocating systems located on the lutoid tonoplast. The opposite pH-dependence in the physiological pH range of the two operating moieties makes this system function as a true bioosmotic pH-stat in the regulation of the cytosol pH in latex. Furthermore, it was shown that the two pH-stats did not work independently but that they could cooperate or at least interfere with each other so that the cytosol pH remained effectively protected against excessive acidification, the latter having been shown to be unfavorable for the latex regeneration mechanism and then for rubber production.

Moreover, as H+ translocation systems were shown to operate electrogenically, they build up and control the magnitude of a transtonoplastic electrochemical proton gradient. Fur-


thermore, the proton motive force (A&H+) has been shown to energize the transport and accumulation of solutes within latex vacuoles; some of these are potent inhibitors of numerous key enzymes of the metabolism.

It was then firmly proposed, in line with the functioning of the two opposing H+-pumps at the lutoid tonoplast, that the vacuolar component of latex plays a double role as a “bioosmotic pH-stat’’ and as an essential “detoxifying trap”, thus controlling homeostasis of the cytosol (Figure 6). This would favor active metabolism within latex cells, resulting in high rubber production. 115.’20

I

.

1

SI.

1.

II,

III.

I

! Physiology \

of Rubber Tree Latex

>

The Laticiferous .Cell and Latex- A Model of Cytoplasm

I

I .

Ed i tors

Jean d'AÜzac Professor

University Montpellier II Montpellier, France

, i Department of Applied Plant Physiology c

Jean-Louis Jacob i Head of Laboratory of Plant Physiology

Montpellier, France IRCA-CIRAD

Department Chief Plant Physiology and Biotechnology

ORSTOM Abidjan, Ivory Coast

CRC Press, Inc. Boca Raton, Florida

Documents

Metabolic regulation and homeostasis in the laticiferous cellhorizon.documentation.ird.fr/exl-doc/pleins_textes/pleins_textes_6/b_fdi_45-46/... · metabolism towards the synthesis