Bicarbonate Fixation and Malate Compartmentation in Relation to … · KHCOO. Experiments were terminated by pouring 25 ml of boiling 80% (v/v) ethanol over the roots, following which

Plant Physiol. (1971) 47, 525-531

Bicarbonate Fixation and Malate Compartmentation in Relationto Salt-induced Stoichiometric Synthesis of Organic Acid'

Received for publication January 12, 1970

BENJAMIN JACOBY2 AND GEORGE G. LATIESDepartment of Botanical Sciences and Molecular Biology Institute, University of California at Los Angeles,Los Angeles, California 90024

ABSTRACT

The relationship of malate synthesis to K absorption fromsolutions of K2SO4 and KHCO3 was compared in nonvacuolatebarley (Hordeum vulgare) root tips and whole excised roots.The comparison has permitted separation of the processwhich evokes organic acid synthesis from that which leadsto stoichiometry between net acid equivalents formed and ex-cess K+ absorbed from K2SO4, on the one hand, and total K+absorbed from KHCOs, on the other. Both in tips and inroots K+ uptake from 20 mN salt solution exceeds malatesynthesis in the first hour. In vacuolate roots the expectedstoichiometry is achieved with time. When root tips are trans-ferred to dilute CaSO4, malate is rapidly metabolized, and K+is lost to the solution. By contrast, in excised whole roots themalate level remains unchanged, the salt-induced organic acidpresumably being retained in the vacuole. In excised rootsmalonate leads to a marked drop in malate levels in untreatedroots as well as in roots which have experienced salt-inducednet malate synthesis. In consequence, it is contended thatmalonate makes available normally sequestered vacuolarmalate.The general hypothesis is offered that the bicarbonate

level of the cytoplasm controls organic acid synthesis byphosphoenolpyruvate carboxylase, and that the cytoplasmicbicarbonate level is raised either by exchange of cytoplasmicHI for external cation, or by bicarbonate absorption directly.Stoichiometry, in turn, is achieved by the accumulation inthe vacuole of the double salt of malate.

The time-honored observation linking organic acid syn-thesis in plant tissue to preferential cation uptake is stillattended by the primordial questions of what elicits synthesis,and why there is a stoichiometric relationship between theequivalents of acid produced and the excess of cation absorbedover the counter anion taken up. It has been amply establishedthat the organic acid synthesis which accompanies excesscation absorption is the result of dark CO, fixation (3, 14,17, 18, 43), and it remains to determine the manner in whichone or more of the multiplicity of carboxylating enzyme sys-

'Supported by Contract AT(11-1)-34 No. 61 award by theUnited States Atomic Energy Commission.

'On leave from: Department of Agricultural Botany, The He-brew University of Jerusalem, Rehovot, Israel.

tems (11, 40, 47) is influenced by the act of preferentialcation uptake, and the reason the response bears such aprovocative quantitative relationship to the causative event.

Rather different explanations have been offered for thephenomenon under consideration. On the one hand, it hasbeen suggested by Burstrom (3), and subsequently by others,that additional organic acid synthesis is directly evoked bywhat is in effect the titration of cytoplasmic organic acid,specifically, the conversion of malic acid to malate. Thetitration may be accomplished by the exchange of cytoplasmicH+ for external cation (15, 17, 18, 43), or by the metabolismof cytoplasmic NO,-, with consequent NH. formation (3).Alternatively, it has been suggested that the signal forenhanced organic acid synthesis is the withdrawal to thevacuole of the organic acid salt formed by H+/ cation ex-change (42). For reasons developed below we have abandonedthe notion that sequestration of organic acid salt in the vacuoleelicits organic acid synthesis. However, we impute the ob-served stoichiometry to vacuolar uptake. Bicarbonate and highexternal pH have both been implicated in organic acid syn-thesis, and the explanation of the influence of each has rangedfrom the direct involvement of bicarbonate (14, 37) to theinfluence of pH per se (32).At salt concentrations in the range of system 2 (20 mM; see

Refs. 9, 21, 41) Torii and Laties (42) observed "4C-bicarbonateincorporation into malate by nonvacuolate root tips of maizeor barley to be much the same in solutions of KCl and K,SO,.By contrast, in agreement with a welter of studies with vacuo-lated roots (10, 12, 17, 18, 43), K&SO, a salt with a poorlyabsorbable anion, evoked pronounced organic acid synthesiscompared with KCl. Since we have taken system 2 to representtonoplast transport (21), we have been prone to emphasize therole of the tonoplast in organic acid synthesis. However, Hiatt(10) has unequivocally demonstrated net organic acid synthesisin response to preferential cation uptake in the system 1 range(up to 0.5 mM), a range in which plasma membrane transportis presumably in kinetic control (21, 41). While overt netsynthesis takes longer at low concentrations, the character-istics of the process are the same as at high external salt levels.In connection with Hiatt's observations it is to be emphasizedthat in absorption periods of 6 hr the bulk of salt absorbed isdelivered to the vacuole, even at low external concentrations(5, 29). Thus, with no further information it might be deducedthat control of organic acid synthesis is related to tonoplasttransport even at salt concentrations in the range of system 1.To resolve the apparent anomaly between the observations

of Torii and Laties (42) and those of Hiatt (10), and to ex-amine more rigorously the question of the relation of vacuola-tion to salt-induced organic acid synthesis, we have comparedsalt-induced organic acid synthesis in nonvacuolate root tipsand vacuolate-excised roots, this time on the basis of net

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Plant Physiol. Vol. 47, 1971

malate synthesis rather than on the incorporation of 'CO2.Our studies have unequivocally implicated preferential cationuptake to the cytoplasm as the cause of salt-induced organicacid synthesis, and at the same time have re-emphasized theinadequacy of 4CO0 incorporation into malate as a quantita-tive criterion of induced synthesis.A comparison of salt-induced organic acid synthesis in tips

and in subapical root tissue has allowed a separation of thesynthetic events from the sequestration process and has demon-strated the independence of synthesis and stoichiometry. Fur-thermore, studies with root tips have shed light on the avail-ability of cytoplasmic malate to mitochondrial metabolism(23).

MATERIAIS AND METHODS

Barley (Hordeum vulgare, var. Sacramento) was grown aspreviously described (41, 42) at 25 C in the dark. Roots wereexcised from 4-day-old seedlings and washed repeatedly indemineralized water before use. Roots or root tips were incu-bated in Erlenmeyer flasks in a reciprocal thermostatic shakerat 25 C. "Roots" will henceforth mean excised roots withtheir tips; "root tips" designate 1.2- to 1.5-mm apical segmentsof excised roots. All incubation media contained 0.4 mN CaSO,in addition to specified addenda.When potassium (abeled with 'Rb) uptake was to be de-

termined, free space potassium was removed by washing theroots, before extraction, in 20 mN CaSO, for four successive1-min periods. Washout was essentially complete in one wash.Free space potassium was removed in the same way beforetransfer of roots to CaSO, following a pulse in K2SO, or

KHCOO.Experiments were terminated by pouring 25 ml of boiling

80% (v/v) ethanol over the roots, following which the rootswere ethanol-extracted for 30 min on a steam table and ex-tracted twice again as previously described (42). The com-bined ethanol extracts were dried at 50 C under partial vac-uum; the dry residue was dissolved in 5.0 ml of 50% (v/v)ethanol and then brought to the desired volume with water.

Incorporation of 14C into organic acids and separation oforganic acids for IC counting as well as for titration were alsocarried out as described before (42). When organic acids were

separated for titration, larger amounts of tissue were em-

ployed, and appropriately larger ion exchange columns were

used for separation. The residues obtained after drying theformic acid eluates from the anion exchange column weredissolved in water, and aliquots were taken for 14C counting or

titration according to the experiment.Potassium was in effect labeled with "Rb, and uptake was

measured by counting of aliquots of the redissolved ethanolextract. Rubidium-86 and 14C were counted in a liquid scintilla-tion counter, with aliquots of sample added to Bray's fluor (2,20). The use of "Rb as a tracer for potassium movement hasrecently been questioned (25). Part of the difficulty lies in theinitial loss of potassium from roots following cutting (31), a

phenomenon which beclouds the meaning of early measure-

ments of isotope absorption. Our excised roots were aeratedin water at least 1 hr before use, a procedure designed to avoidthe foregoing pitfall. With this precaution, and with Cal+ pres-

ent, Laiuchli and Epstein have shown that "Rb uptake is a re-

liable indicator of K absorption (22, 33).Malate was determined in aliquots of the redissolved dried

ethanol extract by measuring NAD reduction in the presence

of malic dehydrogenase (13). In one experiment KHCOO was

double-labeled with "Rb and 14C bicarbonate. Rubidium up-take, 1C incorporation into organic acids, and malic acid weremeasured. Rubidium was retained on a cation exchange col-

umn, while malate and 14C incorporation were measured asusual. Subsequently, 'Rb was eluted from the column with 5N HCl. The eluate was evaporated on a steam table, organiccompounds were digested with HNO., and the digestion mix-ture was dried again. The residue was finally dissolved in water,and aliquots were counted for 'Rb.

Potassium sulfate solutions were adjusted to pH 4.5 withH..SO,. Tris-malonate and potassium malonate at pH 5.0 wereobtained by titrating malonic acid with the respective base.Tris-HCOO was obtained by passing a stream of CO. throughtris base.

RESULTS

The effects of 0.1 mIiN KCI and K4SO, on organic acidsynthesis measured by 1C incorporation and by titration werecompared in a 6-hr experiment. The label incorporated in thetwo cases was not significantly different, whereas net malatesynthesized in K1S0O was five times that in KCI-in agreementwith Hiatt's observations (10). At low external salt concentra-tions, exchange labeling of carboxyl groups or organic acidturnover is too large relative to net fixation for the accuratedetermination of net synthesis by 1'C incorporation (cf. 42).Thus, in low salt, the measurement of 14C incorporation intoorganic acids is not a suitable method for the study of thequantitative relationship between excess cation uptake andorganic acid synthesis (cf. 12). Respiratory dilution of 1COfurther obscures the picture. In all further experiments, netchanges in malate content were measured biochemically (13).

In order to study cytoplasmic changes in organic acids inresponse to preferential cation absorption, the plan was to useshort experimental periods and low external salt concentra-tions, conditions designed to accentuate events in the cyto-plasm (5, 31). However, since salt-induced net malate synthesisproved immeasurably low under such conditions, we turnedour attention to root tips where, because of the absence ofvacuoles, high salt concentrations could be used withoutambiguity arising from the influence of tonoplast transport onorganic acid synthesis. The enhancement of K+ uptake bybicarbonate is accompanied by a marked increase in organicacids (14, 43). In consequence we investigated the effect ofKHCO. on root tips and compared the relative effects of KCIand K,S04. To examine the influence of K1S04 unambiguously,the pH was adjusted to 4.5 in given experiments to precludeany bicarbonate effect. The effect of pH per se is of minorimportance in the range from 4 to 8 (14, 19).The malate content of barley root tips increased consider-

ably during a 1-hr incubation period in either 20 mN KHCOOor KS04; KCI induced only a very small amount of malatesynthesis (Table I). Potassium uptake by the tips from KCI andK,S0, was similar, and about half that from KHC0O. Malateincrease in KHCOO was in turn about double that in K,S04. Inboth K2S0, and KHCO8, the increase in equivalents of malatein the tissue during the 1-hr incubation period was only about40% of the equivalents of potassium absorbed, as measured by"Rb uptake.

Transfer of barley root tips to dilute CaSO, (0.4 mN) subse-quent to a 1-hr 20 mN KHCOs pulse resulted in a rapid de-crease of previously synthesized malate. Almost all of it disap-peared within about 3 hr (Fig. 1). In similar experiments withexcised whole roots (Fig. 1), malate synthesis continued afterthe 1-hr pulse period in 20 mN KHCO8. During the 1st hr aftertransfer to CaSO, malate content increased by approximatelythe same amount as during the pulse period. Also, in contrastto root tips, the amount of malate in intact roots did notchange when net synthesis stopped after the 1st hr in CaSO4,but rather remained almost constant for several hours. These

JACOBY AND LATEES526

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HCO3-FIXATION AND MALATE COMPARTMENTATION

Table I. K (86Rb) Uptake and Malate Synthesis by Root TipsIncubated in Various Potassium Salts

Twenty milligrams of 1.2- to 1.5-mm tips were incubated in 10ml of 20 mN salt solution for 1 hr. Initial malate content = 1.4pAeq/g fresh wt.

Salt in Medium pH K (SRb) Uptake Malate Change

jaeq/gKHCO3 8.5 27.0 +10.8K2SO4 5.9 14.6 +5.7KCI 5.9 13.4 +1.2

0'

0

E

z

w0

cnw

0

xw

z

I-z

wLJ

z

0

w

11 _

10-

9-

8-

7-

6-

5-

4

3-

2-

_

Transfer toCO S04

Roots

0

I

I'

II

I,

I KTI PS

527

well as when transferred 3 hr after the pulse. The constancyof the elevated malate level with time in excised roots in theabsence of malonate is in marked contrast to the rapid disap-pearance of malate in tips once synthesis is terminated (seebelow). The apparent rapid sequestration of malate from themetabolic pool in vacuolated tissue is consistent with the factthat salt-induced malate is largely in the vacuole, as deducedfrom malate efflux studies after as short a time as 30 min (1,30). The malate content of untreated roots also decreased in

malonate by more than 60%. Thus we must deduce that inexcised barley roots either malonate leads to the utilization ofvacuolar malate, or more than half the malate in untreatedroots is in the cytoplasm. The latter presumption is totallyuntenable (26), and, in consequence, the malonate-induceddrop of malate to the initial level in pulsed roots cannot beaccepted as proof that cytoplasmic malate solely and totallycomprises the malate which disappears (cf. 23).

Table II shows the result of an experiment determining thefate of potassium (Rb-labeled), malate, and labeled carbonfixed in organic acids when root tips were transferred to diluteCaSO, after a 1-hr pulse in 'Rb- and SC-labeled KHCOs. Thedisappearance of malate is due to its metabolism and not toefflux, as apparent from its absence in the CaSO, medium.Malate is not transformed to another acid; this is evidentfrom the disappearance of 85% of the radioactivity in the or-

ganic acid fraction. Metabolism of malate in root tips is ac-

companied by efflux of previously absorbed potassium (Rb-labeled).A comparison of the time relations of potassium uptake and

malate synthesis in excised whole roots is presented in TableIII. During a 1-hr period in 20 miN KHCO8 or K&SO4 the ap-

parent amount of potassium absorbed is about double theequivalents of net malate formed. During the 1st hr aftertransfer to dilute CaSO, this discrepancy is compensated byfurther malate synthesis. When roots are incubated continu-ously in 20 mN KHCO. or K,SO for more extended periods,as in most previous experiments of this kind (10, 17, 18, 43),the relative excess of absorbed potassium over the increase inmalate content decreases with incubation time (Fig. 3). Thus in

T2 3 4 5 6 7

HOURS

FiG. 1. The change in malate content of excised roots and ofroot tips during bicarbonate treatment and subsequent incubationin CaSO.. KHCOs, 20 mN; CaSO., 0.4 mN; transfer to CaSO4 at1 hr.

differences between tips and intact excised roots may beascribed to the vacuolation of the latter and seem to indicateseparation of newly synthesized malate from metabolic poolsin excised barley roots. Lips and Beevers (23) concluded thatmalate synthesized in maize roots in a 15-min pulse period re-

mains in the cytoplasm, while the initial malate present in un-

treated roots is in the vacuole. The conclusion is based on theirobservation that malate drops to the initial level when pulsedroots are transferred to malonate. If these assumptions arecorrect, malate should not decrease when fresh, untreatedroots are incubated in malonate. To test this expectation, wecompared the effect of malonate on the fate of malate in un-

treated barley roots with that in roots in which the malate levelwas raised with a KHCOs pulse.

Malate content of barley roots previously pulsed withKHCOS decreased to the prepulse level when transferred to 0.1M tris-malonate at pH 5.0 (Fig. 2). Malonate is effective atconcentrations as low as 30 mm. The decrease occurred whenroots were transferred to malonate 1 hr after the pulse, as

Transfer to

1-

v)0a0

E

I-z

z0

0

w

-J

c

6 -

4-

12 -

10 -

8-

6-

4-

2-

Transfer to /Ca S04

/

/

/*~ K HCo3

Malonate

2 3 4

HOURS

* 0

55 6 7 8

Fio. 2. The effect of malonate on the malate content of un-treated and bicarbonate-pretreated barley roots. KHCO8, 20 MN;CaSO4, 0.4 mN. Tris-malonate, 0.1 M, pH 5.0; 0.2 g of roots.





Table II. K (86Rb) Uptake and Malate Syntthesis by Root Tips duiringa KHCO3 Pulse anid Fate of K and Malate during Further

Incubation in CaSO4Twenty milligrams of tips were incubated for 1 hr in t6Rb- and

'4C-labeled 20 mN KHCO3, 0.4 mN CaSO4, pH 8.5. Some of thesamples were then transferred for further 4 hr to 0.4 MN CaSO4.

Material Analyzed K (B6Rb) Malate 14CAbsorbed eIncorporated

jseq/g in tissue dpinTips after KHCOs pulse 23.0 13.5 50,000Tips after KHCO3 pulse and further 12.4 4.9 7,400CaSO4 incubation

in finalmedsum

CaSO4 medium after incubation 8.8 Trace1 8,300'

l Total counts outside-unidentified.

Table III. Potassium (86Rb) Uptake and Malate Synthesis byExcised Roots during KHCO3 or K2S04 Pulse anid durinig Further

Incubation in Dilute CaSO4Roots, 0.2 g, were incubated for 1 hr in 20 mN KHCO3, pH 8.5,

or K2SO4, pH 4.5, and then transferred to 0.4 mN CaSO4 for 1 hr.

After Pulse Period

Pulse Treatment Malate ChangeK ("6Rb) Malate after CaSO4 Perioduptake change

jieqigKHCO3 11.3 +5.7 +11.5K2SO4 4.4 +2.3 +4.4

KSSO4, stoichiometry is apparently achieved after about 3 hr.In KHCO3 the excess of apparent potassium uptake overmalate synthesis decreased from 60% after the 1st hr to about25% after 2 hr, and to approximately 12% after 4 hr. Thelarge effect of bicarbonate on potassium uptake as well as onmalate synthesis is also demonstrated in Figure 3.

While bicarbonate obviously serves as a reactant in carboxyl-ation by PEP3 carboxylase (see "Discussion"), it may con-ceivably exert an indirect effect as a buffer, serving to removecytoplasmic H ions exchanged for external K+. To examine thepossibility that bicarbonate acts simply by lowering the ex-ternal hydrogen ion level, excised roots were incubated for 1hr in 20 mN K,SO,, pH 4.5, at root to medium ratios of 0.2g/ 10 ml to 0.2 g/ 100 ml. Neither potassium uptake nor malatesynthesis was significantly affected by the variation in mediumto root ratio. Since dilution of extruded H+ has no perceptibleinfluence on K+ uptake or malate synthesis, the effect of bi-carbonate is not attributable to its buffering characteristics(cf. 14).KHCO presumably delivers more bicarbonate (and K+) to

the cytoplasm than is created by K+/H+ exchange in KSSO,solution. Bicarbonate per se is implicated as the causative agentin organic acid synthesis. Tris-carbonate at a concentration of20 mm tris, pH 7.3, was presented to excised roots as an incu-bation medium of high bicarbonate content with a poorlyabsorbable cation. Independent bicarbonate transport mediatedby a bicarbonate pump has been reported (34, 37). Figure 4shows considerable continuous malate synthesis in this mediumin spite of very limited absorption of the tris cation, and muchin excess of it. Malic acid formed in tris-carbonate does not

8Abbreviations: PEP: phosphoenolpyruvate; OAA: oxalacetate.

disappear after transfer of the root to dilute CaSO, (Fig. 5).Malate synthesized under these conditions is apparently re-moved from the metabolic pool in the same manner as malatesynthesized in the presence of K2SO or KHCO,.

DISCUSSION

A comparison of net carboxylation in relatively nonvac-uolate root tips with that in vacuolate sections of barley root inresponse to preferential cation uptake (from K,S04) or to

48

i44j-40 KHCO3

U / MalateX36; /

zu 32- /

z /° 28 /

a /?pz 24-

2 16-z /1J /

'a Mala$ote

4- K2 S04

2 3 4

HOURS

FIG. 3. The course of K+ (C'Rb) uptake and malate synthesis inexcised barley roots. KHCO3, 20 MN; K2SO4, 20 mN, pH 4.5.

H 14z

Z 12-00 Malate /c)/

0-10

<. 8

6-

Z 4- / TRIS- 14C

< 2-

C-_z

1 ~~2 3 4

HOURS

FIG. 4. Malate synthesis and tris uptake by excised barley roots.20 mN "4C-tris-bicarbonate, pH 7.3 (tris-(hydroxymethyl-"C)-aminomethane, New England Nuclear).

528 JACOBY AND LATIES



HCO3-FIXATION AND MALATE COMPARTMENTATION

bicarbonate absorption (from KHCO.) has made it clear thatthe evocation of organic acid synthesis and the normally ob-served stoichiometry between excess cation absorbed and netorganic acid synthesized are separable phenomena. In roottips the signal for synthesis is perforce unrelated to vacuolarsequestration, and by the same token the malate formed in re-sponse to a given salt treatment is rapidly metabolized whensynthesis is curtailed. By contrast, salt-induced organic acidin the cells of proximal root sections is soon transported to thevacuole and becomes relatively unavailable metabolically (cf.23, 26). Vacuolar sequestration accounts both for metabolicunavailability and for stoichiometry-the organic acid beingmoved to the vacuole as the double salt. What evokes netcarboxylation?

Prolonged malate accumulation in vacuolate cells in re-sponse to preferential cation absorption is almost certainly notto be explained in terms of a pH-induced shift in equilibriumin a reaction sequence in which malate is the end product (cf.11). As has been mentioned, the malate of mature root cells ispredominantly sequestered in the vacuole and largely unre-sponsive to metabolic events in the cytoplasm (23, 26). Further,for the purported equilibrium malate level to rise steadily withtime in response to a change in cytoplasmic pH, the lattermust also rise steadily with time. In fact, bicarbonate fixationundoes the transient pH rise evoked by K+/H+ exchange orbicarbonate absorption and tends to keep the pH constant, ifnot to lower it. Finally, in this limited exposition of reasonsfor rejecting the equilibrium concept, there is no basis thereinfor the consistently observed stoichiometry between excesscation uptake and net malate synthesis. Even if stoichiometrywere determined in the cytoplasm, equilibrium considerationsin no way anticipate that a unit decrease in H+ concentrationwill evoke an equivalent increase in malate level.

In our view a rise in cytoplasmic bicarbonate is the primemover in organic acid synthesis whether bicarbonate is createdby an exchange of cytoplasmic Ho for external cation (K+ inthis case), or whether bicarbonate is absorbed from solution assuch. While others have espoused this view primarily on thebasis of experimental observations (14, 16), there are com-pelling logical reasons for coming to the same conclusion (seebelow).

So far as has been reported, three carboxylating enzymeswarrant consideration in regard to organic acid synthesis inplant tissues, and particularly in graminaceous roots (11, 12,40). PEP carboxylase (EC 4.1.1.31) mediates a virtually ir-reversible carboxylation wherein OAA and inorganic phos-phate are products (27, 47). PEP carboxykinase (EC 4. 1. 1. 32)and malic enzyme (L-malate-NADP+ oxidoreductase [decar-boxylating], EC 1 .1 . 1 .40) mediate freely reversible carboxyl-ating reactions (47), the former yielding oxalacetate and ATP(from ADP), and the second yielding malate directly from theNADPH-linked reductive carboxylation of pyruvate. In allcases it is malate which accumulates owing to the prevalenceof cytoplasmic malic dehydrogenase (38).

Freely reversible carboxylating reactions-such as thosemediated by PEP carboxykinase or malic enzyme-will notfavor prolonged net organic acid synthesis which dependsupon the attainment of equilibrium. The assertion is particu-larly true where the enzymes in question coexist with PEPcarboxylase, which mediates the virtually irreversible carboxyl-ation of PEP to OAA. Since the enzymes in question arefound together in barley root cytoplasm (8, 11), carboxyla-tion of PEP by other than PEP carboxylase must have a kineticexplanation. Equilibrium (thermodynamic) considerations perse allow no role for PEP carboxykinase in the presence ofPEP carboxylase. Thus it would seem that PEP carboxy-kinase-and malic enzyme as well-have special functions un-

'4-1

in00

EI-

zw

z0J

w

I--J

12-

10-

8-

6-

4-

2-

Transfer toCa S04

14s1

TRIS bicarbonate

I I

2 3 4HOUR

75 6 7

FIG. 5. The malate content of excised barley roots transferredto CaSO4 following a period in Tris-bicarbonate. 14C-tris-bicarbonate,20 mN, pH 7.3. CaSO4, 0.4 niN.

related to net CO. fixation (i.e., gluconeogenesis; see Ref. 47).The extensive incorporation of labeled CO, into malate in theabsence of net fixation (12, 42) is probably due to the latterenzymes.The question of whether organic acid synthesis engenders

preferential cation absorption or is the consequence thereofmay serve to accentuate the points at issue. It will help at thesame time to query whether CO, or bicarbonate is the reactantspecies. While PEP carboxykinase (4) and malic enzyme (6)fix CO2, HCO.- is the reactant for PEP carboxylase (26, 46).The cytoplasm is bathed in a constant stream of respiratory

CO2. If CO2 were the species fixed, there is no obvious reasonwhy, taking into account sequestration of the product in thevacuole, net organic acid synthesis should not take place con-tinuously until the cell becomes saturated with malate. Nor isthere any reason why synthesis should depend on K+/H+ ex-change. The exchange of cytoplasmic H+ for external K+wouldserve only to titrate cytoplasmic organic acid, and titrationper se offers no basis as a signal for further synthesis. Whilecontinued preferential cation uptake may depend upon K+/H+exchange, and hence upon organic synthesis, synthesis is notin turn dependent on exchange.

Another consideration mitigating against CO2 as the speciesfixed is the high Km for CO2 for both PEP carboxykinase(CO2 + HCO.-, 20 mm [4, cf. 45]) and malic enzyme (3.8 x10-' M, cf. Ref. 6). The interstitial pCO2 of graminaceous rootswill be low, owing to moderate respiration rates, the small rootdiameter, and the loose packing of the cortex cells. Hence thesteady state cytoplasmic CO2 concentration will be well belowthe Km for CO2for the two enzymes in question (for example,1% C02 in the gas phase is in equilibrium with roughly 0.2mM CO2 in solution at 30 C). In consequence an increase inpCO2should enhance CO2 fixation. In fact, net CO2fixation bybarley roots maintained in 10` N salt was found to be thesame in air as in 5% CO2 in air (12). Only when CO2 is ad-ministered at high external pH, where bicarbonate is formed,does CO, have a demonstrable effect (32, 35). Finally, in thepresence of considerable respiratory CO2 the administrationof small quantities of bicarbonate should have little effect oncarboxylation if it is CO2 which is being fixed. As shownherein (and elsewhere [14, 16, 43]), bicarbonate has a pro-found effect.

529-P-Ia-nt Physiol. Vol. 47, 1971




Thus we are led by indirect considerations to the view thatbicarbonate is the significant species involved in salt-evokedcarboxylation. While the Km for HCO- for PEP carboxylaseis quite low (less than 1.0 mM: [16, 27, 45]), the bicarbonatelevel in the cytoplasm can nevertheless be expected to be in arange where PEP carboxylase activity is proportional to bi-carbonate concentration. As mentioned above, the steady statecytoplasmic CO2 concentration must be less than 0.2 mm. Thepredominant buffering agent in the cytoplasm is in all likeli-hood the cytoplasmic protein itself, and more particularly, inthe range pH 6 to 7, the three to four imidazolium histidinegroups per hundred protein amino acids (on the basis ofroughly 9 g of protein per kg root wet weight [24, 36], about2.5 mm histidine). Since the acid pK values of carbonic acid(6.3) and the histidine imidazolium group (6.1) are much thesame, calculations show that the steady state bicarbonate levelwill be in a range where PEP carboxylase responds to bicar-bonate concentration. While there are no direct measurementsof cytoplasmic pH, vital staining studies indicate the cyto-plasmic pH of plant cells to be 7.0 or slightly above (8). Thelow pH of root extracts (10, 43) is almost surely due to thevacuolar contents and gives no indication of the cytoplasmicpH.We may now view K/H+ exchange as a causative event in

salt-induced carboxylation. K+/H+ exchange leads to bicarbon-ate formation in the cytoplasm, which in turn stimulates car-boxylation by PEP carboxylase. To create the double salt ofthe organic acid-specifically of malate-an additional ex-ternal K+ is exchanged for the hydrogen ion which arises whena neutral molecule (triose) is metabolically converted to an acid(phosphoglyceric acid, and hence to PEP) in the normal courseof glycolysis (see Fig. 6). Direct administration of bicarbonatecan be expected to have an effect equal to, or greater than, that

VACUOLE

G

MALATE=+2K5

CYTOPLASM 1SOIL-U TiON

iLU COS E

TRIOSE NAD

-NADH

PGA -+H+.I ,'s

PGA + K5

C02+OH2O-_ H2 C03

HC03-+ H+

PEP-+K+ HC03-+K'

IJOAA=+2 K+

NADH

NAD

Lr

MALATE +2 K+

SO.-

FIG. 6. Schematic representation of organic acid synthesis in re-

sponse to selective cation uptake. Abbreviations: PGA: phospho-glyceric acid; PEP: phosphoenolpyruvic acid; OAA: oxalaceticacid. The net negative charges depicted for PGA, PEP, OAA, andmalate refer to dissociated carboxyl groups. C02 refers to respira-tory CO2.

elicited by preferential cation uptake, and it does. Bicarbonateis readily absorbed and serves as counterion, so that bicarbon-ate actually stimulates K uptake, and preferential cation up-take is not at issue where the bicarbonate salt is involved (14,18, 37). The very fact that bicarbonate uptake matches K+ ab-sorption implicates bicarbonate as the species fixed. Were CO2fixed, cytoplasmic H+ would be exchanged for external K+, andthere would be no cause for bicarbonate uptake. While K+/H5exchange is the prime mover in organic acid synthesis relatedto preferential cation uptake, exchange is in turn sustained byacid synthesis. Without synthesis, the cytoplasmic pH wouldcontinue to rise-presumably beyond physiological levels, withan attendant diminution of exchangeable H+. K+/H+ exchangedoes not provide the basis of the observed stoichiometry, how-ever, since the amount of bicarbonate formed in consequenceof the exchange depends in part on the buffer capacity of thecytoplasm. The transport of the double salt to the vacuole notonly imposes stoichiometry, but in removing K+ from the cy-toplasm further favors K+/H+ exchange. The latter influenceof vacuolar transport is to be seen in the inhibitory effect ofNH,Cl on K+ uptake from potassium bicarbonate, comparedwith the failure of NH4Cl to inhibit KCI absorption (16). WhileKC1 is transportable to the vacuole, as is potassium malate,potassium aspartate (OAA amination product) apparently isnot. Furthermore, malate has been found to be an effective in-hibitor of PEP carboxylase in corn roots (39), so that malatetransport to the vacuole provides yet another indirect positiveinfluence on cytoplasmic malate synthesis.

Since stoichiometry stems from vacuolar transport, and notfrom the uptake process per se, it is easy to see how carboxyla-tion may persist after the tissue is removed from the appro-priate salt. Under conditions where the rate of K+/H+ exchangeexceeds that of carboxylation, bicarbonate builds up to someextent and sustains subsequent carboxylation. Rapid bicarbon-ate absorption will have the same effect. As shown herein, innonvacuolate root tips stoichiometry is not a feature of pref-erential cation uptake from K2SO4, nor of K uptake fromKHCO,. Further, stoichiometry is frequently not observed inshort uptake periods in root sections of vacuolate cells, whilebeing typical of prolonged uptake.

Finally, there is direct evidence of bicarbonate involvement.Hurd has established that the effect of bicarbonate on K up-take is due to bicarbonate per se and not to pH (14). pH has aminimal effect on absorption in the range 5 to 8 (19, 44, cf.14). The direct influence of bicarbonate has been demonstratedby Steward and Preston (36) and by Jackson and Coleman(16) and is evident in the early work of Ulrich (43), and ofPoole and Poel (32). While phosphate at pH 7.5 elicits exten-sive organic acid synthesis (17, 18, 23, 35), phosphate at pH5.0 has no effect (26).Maruyama et al. (27) have directly implicated bicarbonate

as the reactive species in carboxylation mediated by peanutcotyledon PEP carboxylase by noting the distribution of SQ inOAA and inorganic phosphate following the fixation of 10-labeled bicarbonate. In connection with the report that CO, isthe reactant in maize leaves (46) it is noteworthy that Mukerjiand Ting (28) have described three forms of PEP carboxylasein cotton leaf tissue. With the existence of isoenzymes of PEPcarboxylase it becomes urgent to establish separately the reac-tive species for each isoenzyme. Evidence herein favors bicar-bonate involvement in PEP carboxylase-mediated carboxyla-tion in roots (12, 16-18, 42, 43).

LITERATURE CITED

1. BENNETT-CLARK, T. A. AND D. BEXON. 1943. Water relations of plant cells.III. The respiration of plasmolyzed tissues. New Phytol. 42: 65-92.

2. BRAY, G. A. 1960. A simple efficient liquid scintillator for counting aqueoussolutions in a liquid scintillation counter. Anal. Biochem. 1: 279-285.

530 JACOBY AND LATIES

ova4-+



Plant Physiol. Vol. 47, 1971 HCO3-FIXATION AND MALATE COMPARTMENTATION

3. BuRsTRoM, H. 1945. Studies on the buffer system of cells. Ark. Bot. 32:1-18.

4. COOPER, T. G., T. T. TCHEN, H. G. WOOD, aND C. R. BENEDICT. 1968.The carboxylation of phosphoenolpyruvate and pyruvate. I. The activespecies of "COs" utilized by phosphoenolpyruvate carboxykinase, car-

boxytransphosphorylase, and pyruvate carboxylase. J. BioL Chem. 243:3857-83.

5. CRAm, W. J. 1969. Short term influx as a measure of influx across theplaalemmLa. Plant Physiol. 44: 1013-1015.

6. DALIEL, K. AND J. C. LONDESBOROUGH. 1968. The mechanisms of reductivecarboxylation reactions. Carbon dioxide or bicarbonate as substrate ofnicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydro-genase and "malic" enzyme. Biochem. J. 110: 223-230.

7. DANNER, J. AND I. P. TING. 1967. CO2 metabolism in corn roots. II. In-tracellular distribution of enzymes. Plant Physiol. 42: 719-724.

8. DRAWERT, H. 1968. Vitalfarbung und Vitalfluorochromierung pflanzlicherGewebe. Protoplasmatologia II,D3, 516-517.

9. EpsTmN, E. 1966. Dual pattern of ion absorption by plant cells andby plants. Nature 212: 1324-1327.

10. HATT, A. J. 1967. Relationship of cell sap pH to organic acid changeduring ion uptake. Plant PhysioL 42: 294-298.

11. HwrTT, A. J. 1967. Reactions in vitro of enzymes involved in CO2 fixation ac-

companying salt uptake by barley roots. Z. Pflanzenphysiol. 56: 233-245.12. HwITT, A. J. AND S. B. HsuDiucrs. 1967. The role of COs fixation in

accumulation of ions by barley roots. Z. Pflansephysiol. 56: 220-232.13. HOHORsT, A. J. 193. L(-)-Malate determination with malic dehydro-

genase and DPN. In: H. U. Bergmeyer, ed., Methods of EnzymaticAnalysis. Academic Press, New York. pp. 328-332.

14. HURD, R. G. 1958. The effect of pH and bicarbonate ions on the uptakeof salts by disks of red beet. J. Exp. Bot. 9: 159-174.

15. JACKSON, P. C. AiN H. R. ADAms. 1963. Cation-anion balance duringpotassium and sodium absorption by excised barley roots. J. Gen. Phys-iol. 46: 36938f6.

16. JAcKsoN, W. A. AND COLEmAN, N. T. 1959. Fixation of carbon dioxideby plant roots through phosphoenolpyruvate carboxylas. Plant Soil 11:1-16.

17. JACOBsoN, L. 1955. Carbon dioxide fixation and ion absorption in barleyroots. Plant Physiol. 30: 264-269.

18. JACOBsoN, L. AND L. ORDIN. 1954. Organic acid metabolism and ion absorptionin roots. Plant Physiol. 29: 70-75.

19. JACOBsON, L., R. OvRsTREET, R. M. CAaoN, AND J. A. CHAaTAN.1957. The effect of pH and temperature on the absorption of potas-sium and bromide by barley roots. Plant Physiol. 32: 658-662.

20. JACOBY, B. 1967. The effect of the roots on calcium ascent in bean stems.Ann. Bot. N.S. 31: 725-730.

21. LATIES, G. G. 1969. Dual mechanisms of salt uptake in relation to com-partmentation and long distance transport. Annu. Rev. Plant PhysioL20: 89-116.

22. LAucHu, A. AND E. EPsTzIN. 1970. Transport of potassiuim and rubidiumin plant roots. Plant PhysioL 45: 639-41.

23. LiPs, S. H. AND H. BiuvuaS. 196. Compartmentation of organic acidsin corn roots. U. The cytoplasmic pool of malic acid. Plant Physiol. 41:713-717.

24. LONG, C. ed. 1961. Biochemist's Handbook. Van Nostrand Co., Inc., NewYork. pp. 990-991.

25. MAAs, E. V. AND J. E. LEGGET. 1968. Uptake of 9Rb and K by excised maizeroots. Plant Physiol. 43: 2054-2056.

26. MACLENNAN, D. H., H. BEEVERS, AND J. L. HaART E1. 1963. Compart-mentation of acids in plant tissues. Biochem. J. 89: 316-327.

27. MARUYAMA, H., R. L. EASTERDAY, H. C. CHANG, AND M. D. LANE. 1966.The enzymatic carboxylation of phosphoenolpyruvate. 1. Purificationand properties of phosphoenolpyruvate carboxylase. J. Biol. Chem. 241:2405-2412.

28. MUKERn, S. K. AND I. P. TING. Phosphoenolpyruvate carboxylase isoen-zymes: Separation and properties of three forms from cotton leaf tis-sue. Arch. Biochem. Biophys. In press.

29. OSMOND, C. B. AND G. G. LAnIEs. 198. Interpretation of the dual iso-therm for ion absorption in beet tissue. Plant Physiol. 43: 747-755.

30. OsmoND, C. B. AND G. G. LATIEs. 1969. Compartmentation of malate inrelation to ion absorption in beet. Plant Physiol. 44: 7-14.

31. PrrmAN, M. 1969. Adaptation of barley roots to low oxygen supply andits relation to potassium and sodium uptake. Plant Physiol. 44: 1233-1240.

32. POOLE, R. J. AND L. W. POEL. 1965. Carbon dioxide and pH in relationto salt uptake by beetroot tissue. J. Exp. Bot. 16: 4461.

33. RN8s, D. W. 1969. Sodium and potassium absorption by bean stem tis-sue. Plant Physiol. 44: 547-554.

34. RAvEN, J. A. 1970. Exogenous inorganic carbon sources in plant photo-synthesis. Biol. Rev. 45: 167-221.

35. SPLrsvToEssER, W. E. AND H. BzERs. 1964. Acids in storage tissue. Ef-fects of salts and aging. Plant PhysioL 39: 163169.

36. STEwan, F. C. jAN C. PRESTON. 1941. Effects of pH and the com-ponents of bicarbonate and phosphate buffered solutions on the me-tabolism of potato discs and their ability to absorb ions. Plant Physiol.16: 481-519.

37. SuTcLimE, J. F. jAN R. G. HRD. 1961. Active absorption of bicar-bonate ions by vacuolated plant cells. Recent Advan. Bot. 2: 1116-1119.

38. TiNG, I. P. 1968. Malic dehydrogenases in corn root tips. Arch. Biochem.Biophys. 126: 1-7.

39. TING, I. P. 1968. CO2 metabolism in corn roots. III. Inhibition ofP-enolpyruvate carboxylase by L-rualate. Plant Physiol. 43: 1919-1924.

40. TmIG, I. P. AND W. M. DuGGER, JR. 1967. C0C metabolism in corn roots.I. Kinetics of carboxylation and decarboxylation. Plant Physiol. 42:712-718.

41. ToRII, K. AND G. G. LATIES. 1966. Dual mechanism of ion uptake in re-

lation to vacuolation in corn roots. Plant Physiol. 41: 863-870.42. ToRn, K. Aim G. G. LATIEs. 1966. Organic acid synthesis in response

to excess cation absorption in vacuolate and non-vacuolate sections ofcorn and barley roots. Plant Cell Physiol. 7: 395-403.

43. ULRICH, A. 1941. Metabolismn of non-volatile organic acids in excised bar-ley roots as related to cation-anion balance during salt accumulation.Amer. J. Bot. 28: 526537.

44. WAISEL, Y., R. NEUMANN, AND Y. ESHEL. 1966. The nature of the pHeffect on the uptake of rubidium by excised barley roots. PhysioLPlant. 19: 115-121.

45. WALKER, D. A. AND J. M. A. BROWN. 1957. Physiological studies on acidmetabolism. 5. Effects of carbon dioxide concentration on phosphoenol-pyruvic carboxylase activity. Biochem. J. 67: 79-83.

46. WAYGOOD, E. R., R. MACHE, AND C. K. TAN. 1969. Carbon dioxide, thesubstrate for phosphoenolpyruvate carboxylase from leaves of maize.

Can. J. Bot. 47: 1455-1458.47. WOOD, H. G. AND M. F. UTTER. 195. The role of CO2 fixation in me-

tabolism. In P. N. Campbell and G. D. Greville, eds., Essays in Biochemis-try, Vol. 1. Academic Press, New York. pp. 1-27.

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Bicarbonate Fixation and Malate Compartmentation in Relation to … · KHCOO. Experiments were terminated by pouring 25 ml of boiling 80% (v/v) ethanol over the roots, following which