ORGANIC ACID SYNTHESIS IN RESPONSE TO EXCESS CATION ABSORPTION IN VACUOLATE AND NON-VACUOLATE SECTIONS OF CORN AND BARLEY ROOTS

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1 Plant & Cell Physiol., 7 (1966) ORGANIC ACID SYNTHESIS IN RESPONSE TO EXCESS CATION ABSORPTION IN VACUOLATE AND NON-VACUOLATE SECTIONS OF CORN AND BARLEY ROOTS KENJI TORII 1 AND GEORGE G. LATIES Department of Botany and Plant Biochemistry, University of California, Los Angeles, California, U. S. A. (Received March 29, 1966) CO2 fixation into organic acids in tips and proximal sections of both corn and barley roots was studied as a function of the nature and concentration of the salt in the external solution. 2. In comparison with the level of 14 COafixationby vacuolate proximal sections in, incorporation was markedly enhanced in and diminished in CaCh. By contrast, non-vacuolate root tips were indifferent to the type of external salt with respect to 14 CO2 incorporation into organic acids. 3. The effect of salt type on organic acid formation from "CO2 was most pronounced at relatively high concentrations. 4. The conclusion was reached that organic acid synthesis in response to excess cation uptake is the result of cation movement into the vacuole, and that transport into the vacuole is mediated by the low-affinity component of the dual mechanisms involved in ion absorption. When a variety of plant tissues roots of the Gramineae in particular absorb ions from salts in which the cation is more readily taken up than the anion, organic acid is synthesized endogenously in quantities stoichiometrically equivalent to the excess of cation over anion absorbed (1-5). Organic acid synthesis under these conditions is the consequence of CO2 fixation {2, k, 5), and hydrogen ion arising from organic acids so formed is released to the external solution (3). It is an open question as to how absorbing tissue senses preferential cation absorption, and thereby responds by synthesizing organic acid. The mere exchange of cytoplasmic hydrogen ion for external cation, in effect a titration of endogenous acid, should not evoke CO2 fixation. On the other hand, following the removal of organic acid from the cytoplasm, the reestablishment of equilibrium vis-a-vis the cytoplasmic C02-fixing systems would lead to organic acid synthesis. If cation absorbed in excess of anion is delivered to the vacuole in the company of a cytoplasmic organic acid anion, while the associated hydrogen ion passes to the external solution, 1 Present address: Government Forst Experiment Station, Meguro, Tokyo. 395

2 396 K. TORII AND G. G. LATIES Vol. 7 (1966) CO 2 fixation may be expected as a consequence. Linked passage of both ions of a salt into the vacuole has recently been indicated by MACROBBIE {6). In the past years it has been firmly established that the uptake of a given ion proceeds by two mechanisms which operate in concentration ranges three orders of magnitude apart (7, 8, see 9). TORII and LATIES (0) have recently suggested that the system (system 1) which operates at very low concentrations, i. e. the system which has a high affinity for the ion being transported, is located in the plasma membrane, while the system which functions at higher concentrations (system 2), and which therefore has a lower affinity for the given ion, operates at the tonoplast. Under appropriate conditions, cation uptake by system 2 depends on the nature of the counter-ion, uptake of potassium, for example, being more extensive from the chloride than from the sulfate salt (7). Since, in the concentration range of system 2, the nature of the anion influences the extent of organic acid synthesis (1, 2, U, 5), and since, as noted above, it is reasonable to presume that it is the passage into the vacuole of an organic acid salt which results in subsequent CO 2 fixation, the combined influence of the counter-anion on cation uptake and organic acid synthesis has led to the assignment of system 2 to the tonoplast. On the basis of the above line of reasoning it is to be expected that organic acid synthesis in response to preferential cation absorption will take place only in vacuolate tissue. To test this presumption, CO 2 fixation was compared in non-vacuolate root tips and in vacuolate sub-apical root sections, with respect to the influence of the type of salt in the external solution on the extent of organic acid synthesis. MATERIALS AND METHODS Hybrid starchy corn (Zea mays), Hy 2/07, from Illinois Foundation Seeds, Inc., was soaked and germinated as previously described (5). Freshly harvested primary roots of 3-day-old seedlings (dark-grown at 25 ), 9 to 10 cm long, were cut into segments 0 to 2 mm and 2 to 15 mm, measured from the apex. Barley, variety Sacramento, was germinated and grown in the dark for 4 days in the same way as corn. Freshly harvested barley roots were cut into segments 0 to 1.2 mm and 1.2 to 20 mm, measured from the apex. Labelled bicarbonate was prepared essentially as described by AKONOFF (17) and MACDONALD and LATIES (5). Approximately 2 millicuries U CO 2 was liberated from BaCOs (c 30 mc/mmole) under vacuum in one arm of a Y tube with 1.0 ml 10 percent perchloric acid, and trapped in the other arm in sufficient NaOH to absorb all the "COs released. The ph was adjusted to 8.6 with 0.1 N H2SO4, and the volume brought to 10 ml. The specific activity of this stock solution was established by measuring the radioactivity of the BaCC>3 obtained by precipitating an aliquot with 0.1 M BaCU in the presence of 0.1 M KOH.

3 ORGANIC ACID SYNTHESIS IN RESPONSE TO ION ABSORPTION 397 The incorporation of "CO 2 into various root sections over a 3 hour period was investigated as a function of salt type in the external solution. Approximately 0.2 g tissue was placed in 100 ml salt solution adjusted to ph 6.0 with dilute acid or base, followed immediately by 0.5 ml (about 30 ^c) Na 2 "CO 3 solution. Flasks were tightly stoppered and incubated with shaking for 3 hours at 24, after which the contents of each flask were poured into a BUCHNER funnel, and the root material thoroughly rinsed with cold distilled water. Each sample was extracted for 30 minutes with 25 ml 80 percent boiling alcohol. Extraction was repeated three times, the extracts bulked, and the volume reduced to 5 ml under vacuum on a rotary evaporator. The extract was clarified by filtration. The clarified extracts were fractionated by means of ion exchange chromatography. Two columns 15 cm long and 1.2 cm in diameter were joined together, with IR 120 resin in the column above, and IR 45 below. The organic acid fraction was eluted from the IR 45 column with 5 N formic acid. The volume was reduced under vacuum to 1 to 2 ml, more distilled water added, and evaporation continued until all traces of formate were removed. Aliquots of the final concentrated eluate were taken for the determination of radioactivity with a micro-mil window, gas flow counter. The remaining eluate was transferred to Whatman 3 MM paper along a 272 inch strip, and chromatograms were developed with the organic phase of Ti-butanol, 80 percent formic acid, water (1:1:1 v/v) mixture aged 24 hours. Chromatograms were steamed, dried, and sprayed along the edge with 0.04 percent bromphenol blue. The distribution of radioactivity among the various acids was done with a strip-scanner in conjunction with a gas flow detector and chart recorder. The various acid bands were cut out and eluted with water. The acid was titrated with N NaOH with phenolphthalein as indicator. Alternatively acid quantity was estimated by spraying the entire chromatogram with bromphenol blue and determining the color developed by strip-scanning with a densitometer. RESULTS u COi fixation in corn roots The influence of salt type on "CO2 incorporation in root tips and proximal sections of corn roots is described in Tables I and II. In Table I rubidium is the cation, while in Table II potassium is the monovalent cation. Turning first to Table II, vacuolate sub-apical sections respond to salt type in what may be termed the classical way, while non-vacuolate root tips are relatively indifferent to the nature of the salt in the external solution. Compared with u CC-2 fixation by vacuolate tissue in from which cation and anion are absorbed with essentially equal readiness "CO2 incorporation is enhanced by and diminished by CaCl 2. Thus, with a readily absorbable cation, a poorly absorbable anion favors CO 2 incorporation into organic acid, while

4 398 K. TORII AND G. G. LATIES Vol. 7 (1966) TABLE I The incorporation of u COz into organic acids by corn root os a function of tissue type and the nature of the external salt Cation, rubidium. 2 hr incubation period. s: pmoles acid/g fr. wt.; proximal sections: 40-58pmoles acid/g fr. wt. Tissue 0.1 mm 10 mm Salt Rb2SO< Rb2SC-4 RteSO* RbzSCu Ethanol extract cpm X 10~ 4 /g fresh weight Residue Total Organic acids TABLE II The incorporation of u COz into organic acids by corn root as a function of tissue type and the nature of the external salt Monovalent cation, potassium. 2 hr incubation period. u COz in external solution twice that in Table I. Tissue Concentration Concentration 0.2 mm Salt CaCla K2S0* CaCb Ethanol extract cpm X 10~ B /g fresh weight Residue Total Organic acids mm CaCh CaCl

5 ORGANIC ACID SYNTHESIS IN RESPONSE TO ION ABSORPTION 399 TABLE III The incorporation of u COz into organic acids by barley root as a function of tissue type and the nature of the external salt Monovalent cation, rubidium. 2 hr incubation period. Tissue Concentration 0.2 mm 20 mm Salt Rb 2 SO4 CaClz Rb 2 SO4 CaCl 2 RbzSO* CaCl 2 Rb 2 SO4 CaCb Ethanol extract cpm X 10-*/g fresh weight Residue Total Organic acids with a readily absorbable anion, a poorly absorbable cation exerts the opposite effect. The effect of salt type is more pronounced at higher concentrations, as might be expected. That salt type matters even at low concentrations can be ascribed to ion transport into the vacuole when the cytoplasmic concentration is raised in response to the operation of system 1. At low rubidium concentrations (Table I) U CO 2 fixation is related to salt type much as with potassium salts. At high concentrations, however, the effect of salt type is not at all as expected. Rubidium has an effect on metabolism quite apart from its role as an absorbable cation [11, 12) and in this connection different tissues may respond differently to rubidium salts (compare Tables I and III). Where rubidium is used at relatively high concentrations for prolonged periods in absorption studies, as a potassium isotope in effect, its metabolic influence should be separately examined. It is noteworthy that in corn roots the bulk of the radioactivity is found not in malate, but rather in an acid which chromatographs close to succinate and which appears to be trans-aconitic acid (cf. 10). u COz fixation in barley roots Barley roots evince a generally higher level of CO2fixation than do corn roots. Furthermore, the response to external salt is more pronounced in

6 400 K. TORII AND G. G. LATIES Vol. 7 (1966) TABLE IV The incorporation of u CCh into organic acids by barley root as a function of tissue type and the nature of the external salt Monovalent cation, potassium. 2hr incubation period. sections: 22-29//moles acid/g fr. wt. Tissue Concentration 0.2 mil 20 mm Salt H 2 O CaClz CaSO4 H 2 O CaCk CaSO. H 2 O CaCl 2 CaSO4 H2O K 2 SO4 CaClz CaSO4 Ethanol extract cpm X 10~ 4 /g fresh weight Residue Total Organic acids barley. Table III compares the influence of salt type where the monovalent cation is rubidium. In Table IV, the monovalent cation is potassium. Vacuolate sub-apical tissue is markedly responsive to the nature of the external salt in what may be considered the expected way. By contrast, tips are indifferent to the nature of the external salt. Again, the response to salt type is most evident at high concentration. As with corn, potassium salts are more effective than rubidium salts in evoking the noted response. With barley, the organic acid containing the major part of the label is malate.

7 ORGANIC ACID SYNTHESIS IN RESPONSE TO ION ABSORPTION 401 DISCUSSION In the mineral nutrition literature, ion absorption studies are replete with examples of the influence of the counter-ion on cation uptake [1, IS). In particular, monovalent cation absorption is usually more pronounced from chloride than from sulfate or phosphate salts (2, 7). Nevertheless there are instances where cation uptake is indifferent to the counter-ion (3) and it has been proposed (9) that the noted disparity relates to the capacity of the absorbing tissue for organic acid synthesis by dark CO 2 fixation. When a cation is absorbed in excess of its counter-ion, a stoichiometric quantity of hydrogen ion is formed by CO 2 fixation into organic acid, and the hydrogen ion so formed passes into the external solution (IS). It has been suggested (9) that when CO2 fixation occurs readily in response to excess cation uptake, the rate of cation absorption is unimpaired by the presence of a slowly absorbable counter-ion. Conversely, when CO2 fixation cannot keep pace with the potential cation influx, cation absorption will be slowed by a sluggishly absorbed counter-anion. Tissues vary in their capacity for CO2 fixation (cf. Tables II and IV) and it is to be anticipated that an inverse relationship will be found between CO2 fixation capacity of a tissue and the negative influence of slowly absorbable anions on monovalent cation uptake. Apropos of the foregoing contention, "CO 2 fixation at room temperature in aged potato slices, where ion uptake and metabolism are vigorous, responds to external salt in the expected manner, while 14 CO2 incorporation in the cold where metabolism is at a low ebb in all cases is unaffected by the nature of the external salt (5). Furthermore when metabolically inactive fresh potato slices are compared with metabolically active aged slices with respect to their capacity for chloride absorption from solution, the absorption isotherm for fresh tissue suggests an interdependence of cation and anion absorption, while the isotherm for aged tissue does not. A capacity for organic acid synthesis by aged slices was offered as one explanation for the difference (1U). Although the repository for organic acids synthesized in response to excess cation uptake has not been established, the considerable quantity of acid so synthesized (-4), and the means proposed above whereby the cell may sense excess cation absorption, both suggest that organic acids are stored in the vacuole. It would therefore appear that the low-affinity (high Ks) system of EPSTEIN (7), in being sensitive to the nature of the counter-anion, is associated with compensatory organic acid synthesis during excess cation absorption, and is therefore to be placed at the tonoplast (9). The high affinity (low Ks) system which is indifferent to the counter-ion and which has been assigned to the plasma membrane should have little to do with compensatory organic acid synthesis. On the basis of the foregoing presumptions, tips and sub-apical sections of both corn and barley roots have been examined for the influence of the

8 402 K. TORII AND G. G. LATIES Vol. 7 (1966) type of external salt on the incorporation of "CO2 into organic acids in these tissues. The underlying rationale has been that in non-vacuolate root tips, where only system 1 operates (9) CO2 fixation should be independent of the nature of the external salt, whereas in proximal vacuolate tissue, where both systems operate, CO2 fixation may be expected to reflect the nature of the external salt. Independent cation movement into the cytoplasm in all probability also involves exchange with H + ion. However, in the latter instance cytoplasmic organic acid is in effect titrated, and not removed from the cytoplasm. Furthermore, but a small amount of exchange is involved compared with that which accompanies extensive excess cation absorption into the vacuole. For the foregoing reasons cytoplasmic cation uptake is independent of the counter-ion. It is recognized that the absence of vacuoles in the first 1.8 mm of root tip is not total (15, 16) and that the root apex must differ from sub-apical tissue in ways which transcend vacuolation. The validity of the distinction between tips and sub-apical tissue with respect to the assumptions must therefore depend on the results. Tables II and IV most effectively verify predictions. Thus in both corn and barley U CO 2 incorporation into organic acids in root tips is virtually indifferent to the nature of the external salt compared with the situation in the vacuolate proximal sections. In the latter the sulfate salt evokes a sharp enhancement of "COa fixation compared with that in tissue bathed in, while CaCh brings about a sharp drop in incorporation. In accordance with the contention that system 2 is related to the acid producing mechanism, the influence of the external salt is greatest in the high concentration range. Some response to salt type may be expected in tips since they are not totally free of vacuoles. Some response to salt type may also be expected at low concentrations in proximal sections since accumulation by system 1 is followed by transport across the tonoplast by system 2. The results with barley are more emphatic than with corn, and potassium salts more clearly demonstrate the phenomenon than do rubidium salts. Interestingly, in distinction to barley roots, where malate is the predominant acid, and where malate is labelled most extensively by dark CO 2 fixation, trans-aconitic acid is the most rapidly and extensively labelled acid in corn roots under the same conditions (cf. 10). Further elucidation of this matter will appear separately. Taken together the data of Tables I through IV affirm the hypothesis that "CO2 fixation into organic acids in response to excess cation absorption is a consequence of cation delivery into the vacuole, the latter being implemented by the low-affinity, high Ks, transport system. This work was generously supportd by a grant from the U.S. Atomic Energy Commission. We wish to thank Dr. L. JACOBSON, University of California, Berkeley, who kindly provided the Sacramento barley seed.

9 ORGANIC ACID SYNTHESIS IN RESPONSE TO ION ABSORPTION 403 REFERENCES ( 1 ) A. ULRICH Metabolism of non-volatile organic acids in excised barley roots as related to cation-anion balance during salt accumulation. Am. J. Bot., 28, ( 2 ) L. JACOBSON Carbon dioxide fixation and ion absorption in barley roots. Plant Physiol., 30, ( 3 ) P. C. JACKSON and H. R. ADAMS Cation-anion balance during potassium and sodium absorption by barley roots. J. Gen. Physiol., 46, (U ) W. E. SPLITTSTOESSER and H. BEEVERS Acids in storage tissues. Effects of salts and aging. Plant Physiol., 39, ( 5 ) MACDONALD and G. G. LATIES A comparative study of the influence of salt type and concentration on 14 CO2 fixation in potato slices at 25 C and 0 C. J. Exptl. Bot., 15, ( 6) E. A. MACROBBIE Factors affecting the fluxes of potassium and chloride ions in Nitella translucens. J. Gen. Physiol., 47, (7) E. EPSTEIN, D. W. RAINS and 0. E. ELZAM Resolution of dual mechanisms of potassium absorption by barley roots. Proc. Natl. Acad. Set., U. S., 49, ( 8 ) E. EPSTEIN and D. W. RAINS Carrier-mediated cation transport in barley roots: Kinetic evidence for a spectrum of active sites, ibid., 53, ( 9 ) K. TORII and G. LATIES Dual mechanisms of ion uptake in relation to vacuolation in corn roots. Plant Physiol., 41, (10) D. H. MACLENNAN Compartmentation of organic acids in plant tissues. Ph. D. Thesis, Purdue University. University Microfilms, Ann Arbor, Michigan, (11) J. B. HANSON and J. BONNER The nature of the lag period in auxin-induced water uptake. Am. J. Bot., 42, (12) G. G. LATIES The development and control of coexisting respiratory systems in slices of chicory root. Arch. Biochem. Biophys., 79, (IS) D. R. HOAGLAND Lectures on the Inorganic Nutrition of Plants. Chronica Botanica Co., Waltham, Mass. (14) G. G. LATIES, I. R. MACDONALD and J. DAINTY Influence of the counter-ion on the absorption isotherm for chloride at low temperature. Plant Physiol., 39, (15) R. BROWN and P. M. CARTWRIGHT The absorption of potassium by cells in the apex of the root. J. Exptl. Bot., 4, (16) W. G. WHALEY, H. H. MOLLENHAUER and J. H. LEECH The ultrastructure of the meristematic cell. Am. J. Bot., 47, (17) S. ARONOFP Techniques of Radio Biochemistry, p. 71. The Iowa State College Press.

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