Planta

Planta (1981) 152:74-78

9 Springer-Verlag 1981

Extent of intracellular pH changes during H + extrusion by maize root-tip ceils Justin K . M . R o b e r t s 1, Peter M. R a y 1, N o r m a W a d e - J a r d e t z k y 2, a n d Oleg J a r d e t z k y 2 1 Department of Biological Sciences and 2 Stanford Magnetic Resonance Laboratory, Stanford University, Stanford, CA 94305, USA

Abstract. S i p - N u c l e a r - m a g n e t i c - r e s o n a n c e s p e c t r a o f m a i z e (Zea mays L.) r o o t tips, t h a t h a d been i n d u c e d to e x t r u d e large a m o u n t s o f H § in r e s p o n s e to fusicoccin (FC) in the presence o f p o t a s s i u m salts, indicate t h a t the c y t o p l a s m i c p H does n o t b e c o m e h i g h e r t h a n t h a t o f controls. In fact, the c y t o p l a s m i c p H m a y b e c o m e slightly ( a p p r o x . 0. l p H unit) l o w e r in cells e x t r u d i n g H § E s t i m a t i o n s o f the buffer c a p a c i t y o f the cells s h o w t h a t w i t h o u t active i n t r a c e l l u l a r p H regulation, H + e x t r u s i o n c a u s e d by F C w o u l d cause the i n t r a c e l l n l a r p H to rise by at least 0.6 p H unit h - 1. O u r results indicate t h a t i n t r a c e l l u l a r p H is tightly r e g u l a t e d even d u r i n g e x t r e m e rates o f a c i d extrusion, a n d t h a t a rise in c y t o p l a s m i c p H is n o t the signal linking H + e x t r u s i o n with e n h a n c e d o r g a n i c a c i d synthesis o r o t h e r i n t r a c e l l u l a r r e s p o n s e s to H + pumping. K e y words: C y t o p l a s m i c p H - F u s i c o c c i n - H y d r o gen-ion extrusion - p H - s t a t - R o o t - Zea.

Introduction P l a n t cells e x t r u d e H + when u p t a k e o f c a t i o n s exceeds t h a t o f a n i o n s ( J a c k s o n a n d A d a m s 1963), a n d synthesize e q u i v a l e n t q u a n t i t i e s o f o r g a n i c acids ( U l r i c h 1941). A m o n g the p o s s i b l e signals for t r i g g e r i n g org a n i c - a c i d synthesis d u r i n g H + e x t r u s i o n (see reviews by O s m o n d 1976; S m i t h a n d R a v e n 1979) is s i m p l y a rise in p H o f the c y t o p l a s m due to r e m o v a l o f H + f r o m it ( H i a t t 1967; see also review by D a v i e s 1973), This m e c h a n i s m has been f a v o r e d in recent c o n s i d e r a t i o n s o f the p r o b l e m ( S m i t h a n d R a v e n 1979; D a v i e s 1979), but n o t d e m o n s t r a t e d . A cytop l a s m i c p H rise has also been suggested ( S m i t h a n d

Abbreviations: FC = fusicoccin; Pi = inorganic phosphate; NMR = nuclear magnetic resonance; 6-chemical shift; MDP=methylene diphosphonic acid 0032-0935/81/0152/0074/$01.00

R a v e n 1979; D a v i e s 1979; M a r r 6 1979) to signal int r a c e l l u l a r processes i n d u c e d by i n d o l e a c e t i c acid, a u x i n - t y p e herbicides, a n d the p h y t o t o x i n fusicoccin (FC), which s t i m u l a t e H + e x t r u s i o n (see reviews b y R a y l e a n d C l e l a n d 1977; M a r r 6 1979). W e have s h o w n ( R o b e r t s et al. 1980) t h a t the 3tp_ NMR method of intracellular pH determination ( M o o n a n d R i c h a r d s 1973; Burt et al. 1979) c a n be a p p l i e d to m a i z e r o o t tips. W e r e p o r t here m e a s u r e ments of cytoplasmic and vacuolar pH of maize roottip cells i n d u c e d to e x t r u d e large q u a n t i t i e s o f H + by u n b a l a n c e d i o n u p t a k e a n d by F C .

Material and methods Maize (Zea mays L.) hybrid WW x Br 38 (Customaize Research, Decatur, Ill. USA) were grown for 2 d in the dark, and root tips were harvested as described previously (Roberts et al. 1980). Root tips (ca. 1 mg each, 2.5 g per sample) were rinsed with ice-cold 10 mM glucose with 0.5 mM CaSO4, and placed for various periods in an incubation medium [50 mM glucose, 0.5 mm CaSO4, 2 mM citric acid-l,4-piperazinediethanesulfonic acid (PIPES) brought to ca. pH 6 with tris(hydromethyl)aminomethane (Tris) base with additions as indicated in Results. The solutions were bubbled with oxygen, and were mixed further using a shaker or magnetic stirrer. After incubation, the medium was back-titrated to its initial pH with standardized NaOH to determine H + extrusion, either before removal of the root tips (samples B, C, D and J in Fig. 1) or after the root tips were removed (samples E-I in Fig. 1). Most of the incubation medium was removed before root tips were placed in the spectrometer. The samples were chilled on ice and nuclearmagnetic-resonance (NMR) spectra were obtained at 5~ in 10 or 15 rain at 40.5 MHz, accumulating scans every 0.6 or 0.9 s, in an extensively modified system consisting of a Varian Associates (Palo Alto, Cal., USA) XL-100 spectrometer - equipped with a Nicolet Magnetics (Mountain View, Cal., USA) multinuclear probe and probe hardware, and using a Nicolet Instrument Corporation (Madison, Wis., USA) 1,180 computer. A 10-ram tube containing the tissue sample was placed in a 12-ram tube containing DzO, allowing field-frequency locking. Chemical shifts [6, where 6 = (v~Vref)x 106/Vrer,in which vs and Vrefare the absolute resonance frequencies of a particular sample peak and the reference peak, respectively] are referenced to 0.5 M methylene diphosphonic acid (MDP) in pH 8.9 Tris buffer, located in a coaxial capillary tube.

J.K.M. Roberts et al. : H + extrusion and intracellular pH in maize root-tip cells Aqueous homogenates were made by mortar-grinding 1 g washed root tips in 10 ml ice-cold distilled water for 5 rain; the sample was not filtered or centrifuged. Extracts with 80% boiling ethanol were made by standard procedures (Bassham and Calvin 1957), as were extracts with ice-cold perchioric acid (Abdul-Baki and Ray 1971).

Results F i g u r e 1 B - D shows N M R spectra in the 31p frequency region obtained from maize root-tip samples i n c u b a t e d in buffer a n d 5 m M KC1, with or w i t h o u t 3 - 1 0 - s M F C , for the times indicated. T h e r e are t h r e e major peaks distinguishable: peak 1 (glucose-6-phosphate), p e a k 2 ( c y t o p l a s m i c Pi) a n d p e a k 3 ( v a c u o l a r

i

E

75

P~) r e a s o n s for a s s i g n m e n t s are given in R o b e r t s et al. (1980). T a b l e 1 lists the a m o u n t s o f H § e x t r u d e d into the i n c u b a t i o n by the v a r i o u s samples, t o g e t h e r with the 3's o f p e a k s 1-3 o f e a c h sample. T a b l e 1 also gives p H values c o r r e s p o n d i n g to the m e a s u r e d 6%, these being o b t a i n e d using t i t r a t i o n curves for Pi a n d g l u c o s e - 6 - p h o s p h a t e (see Fig. 1 o f R o b e r t s et al. 1980). It is clear t h a t F C causes no shift in the p o s i t i o n s o f p e a k s 1-3 t o w a r d the reference c o m p o u n d ( M D P ) as w o u l d be o b s e r v e d ( R o b e r t s et al. 1980) if F C c a u s e d the c y t o p l a s m i c or v a c u o l a r p H to rise. F i g u r e 1 A shows t h a t t r e a t m e n t o f r o o t tips with 35 m M N H 4 O H for 5 m i n causes a d r a m a t i c c h a n g e in the ~5 values o f p e a k s 1-3 (indicative o f -

/OOm/n

K2504/FC "r K2SO4/FCpH 5

zo rain K2S@FC

lh K2.qO4/FC pH6.1

30 rn~n KzSO4/FC

I in I h

0

-20

-,~0

o

-~

-4o

CONTROL pH 4.S

CONTROL

-20

-40

C

Fig. IA-K. 40.5 MHz 3~p-NMR spectra of maize root tips incubated in the following media for the indicated lengths of time: A 10 ml 0.5 mM CaSO4, 50 mM gluocse and 35 mM NH4OH, 5 min without bubbling; B incubation medium (see Material and Methods) +5 mM KCI+3.10 -s M FC, 1 h; C same as B, but 15 min; D incubation medium +5 mM KC1, 1 h; E incubation medium +25 mM K2SO4+3. l0 s M FC, 100 min; F same as E, but 70 rain; G same as E, but 30 rain (E-G are spectra from a single tissue sample); H incubation medium only, pH6.1, 1 h; I same as E, but 1 h; J same as I but medium back4itrated to pH6.l after 1 h, before obtaining spectrum; K incubation medium only, but adjusted to pH 4.9 before incubation, 1 h. Peaks 1, 2 and 3 are cytoplasmic glucose-6phosphate, cytoplasmic P~ and vacuolar P~, respectively. The peak at 0 ppm is that of the reference compound, MDP

76

J.K.M. Roberts et al. : H + extrusion and intracellular p H in maize root-tip cells

Table 1. Effect of fusicoccin, KC1 a n d KzSO~ on H + extrusion by maize root tips, chemical shifts (c~'s) o f maize root tip 31p nuclear magnetic resonances, and corresponding p H values. Samples as designated in Fig. 1; p H values given are determined from measured values using titration curves of glucose-6-phosphate (for peak 1) and Pi (for peaks 2 and 3) Sample

A D C B H G F E K J 1

Treatment

5 min 35 m M N H 4 O H 1 h KC1 15 rain KC1/FC 1 h KC1/FC 1 h buffer 30 min K2SO4/FC 70 rain K2SO4/FC 100 rain K2SO4/FC 1 h buffer 1 h K2SO4/FC 1 h K2SO4/FC

Final p H of incubation medium

Acid extruded peq (g fr.wt.) 1

9.2 6.0 b 5.92 b 5.4 b 6.11 5.6 5.38 5.48 4.9 5.04 b 5.0

1.0 1.5 6.0 0 4.6 10.2 15.7 0 9.5 9.8

-6(ppm)

Corresponding p H values

Peak 1

Peak 2

Peak 3

12.25 12.69 12.67 12.68 12.73 12.75 12.81 12.75 12.68 12.7 12.73

13.5 14.70 14.70 14.71 14.70 14.70 14.78 14.64 14.72 14.71 14.79

14.4 16.41 16.40 16.40 16.41 16.39 16.37 16.41 16.43 16.41 16.40

Peak 1 > 8~ 7.16 7.19 7.17 7.12 7.10 7.00 7.10 7.19 7.15 7.12

Peak 2 > 8" 7.20 7.20 7.19 7.20 7.20 7.12 7.27 7.17 7.19 7.11

Peak 3 7.60 5.60 5.60 5.60 5.60 5.60 5.66 5.60 5.57 5.60 5.60

a Technique is not accurate in this p H range b Samples titrated back to p H 6.1 (root tips present) before spectrum obtained

a cytoplasmic and vacuolar pH rise > 1 pH unit). We have previously reported (Roberts et al. 1980) that treatment of root tips with more dilute NH4OH solutions (6 and 12 mM) for a longer period (30 min) induces much smaller changes in the ~5 values of peaks 1-3. These experiments demonstrate that a rise in cytoplasmic and vacuolar pH can be detected when it occurs. Maximum H § efflux from root tips occurs with 25 mM K2SO4 and 3.10 -5 M FC (Lado et al. 1976; Table 1). Figure 1E-K, showing spectra from K2SO4/ FC-treated tissue, indicates no rise in cytoplasmic or vacuolar pH; some of the spectra actually indicate a slight decrease in cytoplasmic pH (see Table 1). Since in the foregoing experiments H + extrusion acidified the medium bathing the root tips, we checked whether an acidic external pH might lower intracellular pH values, offsetting an intracellular pH rise due to H § pumping. Figure 11 shows the spectrum for tissue in incubation medium that had reached pH 5.0 by H § extrusion. Figure 1J shows a spectrum of a similar sample, except that the incubation medium was titrated back to the initial pH value, 6.1, before obtaining the spectrum. No effect of an acidic external pH is apparent (Table 1). The intracellular pH also did not fall if tissue without K2SO4 or FC (not extruding H § was kept in a medium of pH 4.9 (Fig. 1 K, Table 1). All spectra show a large glucose-6-phosphate pool relative to cytoplasmic P~, indicating aerobic conditions (Roberts et al. 1980). Differences in the size of peaks 1 and 2 relative to peak 3 reflect differences in the average length of root tips in a given sample (Roberts et al. 1980), longer root tips containing more vacuolate cells and hence a relatively larger peak 3.

Table 2. Buffer capacity o f maize root tip tissue. Buffer capacities, from potentiometric titration of tissue extracts, are given in geq H + per g fresh weight, per p H unit Sample

p H 5-6

pH 6 7

p H 7-8

A B C

A q u e o u s homogenate HC104 extract 80% ethanol extract

21 18 7

20 14 6.5

18 11 6.5

D

Cytoplasm (calculated m a x i m u m ) a

10

12.5

13.5

a Buffer capacity of water extract (A), minus difference between B and C (apparent buffer capacity of root cap slime)

Buffer capacities of various kinds of root-tip extracts are given in Table 2. The aqueous homogenate contains all buffering substances in the tissue, including intracellular proteins and metabolites as well as extracellular acidic polysaccharides in the cell walls and root-cap slime or mucigel. Slime but not protein should occur in the perchloric-acid extract (line B), whereas slime should be absent from ethanol extracts (line C), which should include only small metabolites. Therefore, buffering due to extracellular slime can be estimated by subtracting C from B, and intracellular buffer capacity can be estimated by subtracting this difference from the total tissue buffer capacity (line A), giving a value of about 14 geq H + (g tissue) 1 (pH unit)-1 around pH 7 (line D). This represents an upper limit for cytoplasmic buffer capacity, since some part of the buffering substances (amino acids, organic acids and Pi) occurs in the vacuoles (Matile 1978; Roberts etal. 1980). Also, this method does not correct for the buffer capacity of cell walls, which occur in extract A, but this is probably a significant

J.K.M. Roberts et al. : H + extrusion and intracellular pH in maize root-tip cells

error only below pH 5.5. The value we deduce for maximum cytoplasmic buffer capacity agrees well with that suggested by Raven and Smith (1976). The values in line C of Table 2 represent the apparent maximum buffer capacity of the vacuoles, if most of the small-molecular weight buffering substances (but not protein) were located therein, in which case the cytoplasmic buffer capacity would be correspondingly less. Discussion

From the H § extruded by FC-treated tissue (Table 1) and estimates of intracellular buffering capacity (Table 2) it is clear that without some kind of internal pH regulation a substantial rise in cytoplasmic and/or vacuolar pH ( > 0.6 pH unit h- 1) would occur during K~SO~- and FC-induced H + extrusion. The 31p_ N M R spectra show that no such rise occurs, and indicate instead that the cytoplasmic pH may be slightly lower in cells extruding large amounts of H +. However, the apparent drop (approx. 0.1 pH unit) is close to the limit of resolution of the N M R technique. Certainly, cytoplasmic pH is closely regulated in plant cells during H + efflux, and the cells evidently do not use acid present in their vacuoles as a source of H § to be extruded. Our results are evidence against the hypothesis that rise in cytoplasmic pH during H § extrusion causes accelerated organic-acid synthesis (the "biochemical pH-stat" hypothesis, Smith and Raven 1979). Cytoplasmic pH of the alga Hydrodictyon, when grown in different nutrient regimes, was also found not to change (Raven and De Michelis 1979; 1980a, b) in the direction anticipated by the "pHstat" model (Smith and Raven 1979). Data of Johnson and Rayle (1976) suggest that organic-acid synthesis is accelerated within 1-2 min of exposure of Arena coleoptiles to FC. Using maize root tips we have found a stimulation of 1"CO2 incorporation into organic acids within 1 min, results which will be reported elsewhere. Within this brief period, the cytoplasmic pH could not rise more than 0.01 pH units by H § pumping, a pH change much too small to activate organic-acid synthesis (Davies 1973). We suggest that some signal other than a cytoplasmic pH rise induces intracellular responses during excess cation uptake, FC action, and, perhaps, auxin action. Alternative possible controls of organic-acid synthesis that have been suggested include cation movement into the vacuole (Torii and Laties 1966), and transfer to the vacuole of cytoplasmic malate (Osmond 1976) which inhibits phosphoenolpyruvate (PEP) carboxylase.

77

The fact that regulation of intracellular pH in response to H § extrusion is so immediate indicates that it might actually occur in response to H + efflux itself. One way the pH regulating mechanism might be controlled is by changes in the cell's membrane potential, which is sensitive to H § efflux because the H + efflux pump appears to be electrogenic (Marr~ 1979). However, while FC-induced H + efflux hyperpolarizes (Marrd 1979), excess cation uptake should tend to depolarize (thus stimulating H § pumping), so it seems unlikely that a change in membrane potential triggers organic-acid production in both cases. Another possibility is that action of the H + efflux pump stimulates organic-acid production. One way this could occur would be if the H + efflux pump caused H+/CO2 antiport. This would initially supply H + intracellularly by dissociation of HzCO3 at the relatively high pH of the cytoplasm, and would build up the cytoplasmic HCO3 concentration, leading to accelerated organic-acid synthesis. Control of organic-acid synthesis by cytoplasmic H C O 3 w a s proposed by Jacoby and Laties (1971), and Johnson and Rayle (1976) reported an immediate stimulation by FC of CO2 uptake by Arena coleoptile tissue. We are currently investigating this possibility, but at present none of the suggested alternative control mechanisms can be excluded by experimental evidence. The many hours of help in harvesting root tips given by our friends and colleagues must be gratefully acknowledged. This research was supported by National Science Foundation grants PCM7809230, PCM7807930, and GP23633, and National Institutes of Health grant RR00711.

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J.K.M. Roberts et al. : H + extrusion and intracellular pH in maize root-tip cells

Marr6, E. (1979) Fusicoccin: a tool in plant physiology. Annu. Rev. Plant Physiol. 30, 273-288 Matile, Ph. (1978) Biochemistry and function of vacuoles. Annu. Rev. Plant Physiol. 29, 193-213 Moon, R.B., Richards, J.H. (1973) Determination of intracellular pH by 31p magnetic resonance. J. Biol. Chem. 248, 7276-7278 Osmond, C.B. (1976) Ion absorption and carbon metabolism in cells of higher plants. In: Encyclopedia of plant physiology, [N.S.I, vol. 2A, pp. 347-372, Lfittge, U., Pitman, M.G., eds., Springer, Berlin Raven, J.A., Smith, F.A. (1976) Cytoplasmic pH regulation and electrogenic H + extrusion. Curr. Adv. Plant Sci. 8, 649~660 Raven, J.A., De Michelis, M.I. (1979) Acid-base regulation during nitrate assimilation in Hydrodictyon africanum. Plant, Cell and Environment 2, 245-257 Raven, J.A., De Michelis, M.I. (1980a) Acid-base regulation during ammonium assimilation in Hydrodictyon africanum. Plant, Cell and Environment 3, 325-338 Raven, J.A., De Michelis, M.I. (1980b) Acid-base regulation dur-

ing ammonium and nitrate assimilation in Hydrodictyon africanurn. In: Plant Membrane Transport, 579-600, Spanswich, R.M., Lucas, W.J., Dainty, J., eds. Elsevier/North-Holland, Amsterdam Rayle, D.L., Cleland, R.E. (1977) Control of plant cell enlargement by hydrogen ions. Curr. Top. Dev. Biol. 11, 187-214 Roberts, J.K.M., Ray, P.M., Wade-Jardetzky, N., Jardetzky, O. (1980) Estimation of cytoplasmic and vacuolar pH in higher plant cells by s~p NMR. Nature (London) 283, 870-872 Smith, F.A., Raven, J.A. (1979) Intracellular pH and its regulation. Annu. Rev. Plant Physiol. 30, 289-3 l I Torii, K., Laties, G.G. (1966) Organic acid synthesis in response to excess cation absorption in vacuolate and non-vacuolate sections of corn and barley roots. Plant Cell Physiol. 7 395403 Ulrich, A. (1941) Metabolism of non-volatile organic acids in excised barley roots as related to cation-anion balance during salt accumulation. Am. J, Bot. 28, 526-537 Received 24 November; accepted 30 December 1980