Planta

Planta 135, 297-299 (1977)

9 by Springer-Verlag 1977

Proton Co-transport of Sugars in Phloem Loading F. Malek and D.A. Baker School of Biological Sciences, University of Sussex, Brighton, BN1 9QG, U.K.

Abstract. Loading of 14C-labelled sugars from the hollow petiole of R i c i n u s c o m m u n i s L. was stimulated by potassium and by low pH in that both the 14Cactivity and the sugar concentration of phloem sap collected from a nearby incision increased. A pH drop was observed in the solution perfusing a hollow petiole. This pH drop was greater in the presence of potassium and less in the presence of sugars, while the uncoupler CCCP induced a pH rise in the perfusing solution. Sugars were detected in the perfusing solution when it was buffered at pH > 9. A model is proposed for a proton co-transport of sugars from the free space driven by a linked proton efflux/potassium influx pump.

Key words: Phloem-loading Ricinus Sugar transport.

Materials and Methods 2% solutions of l~C-labelled sugars (1 I~Ci ml 1) in phosphate buffer were perfused through the hollow petiole of mature attached leaves of Rieinus comrnunis L., the leaf lamina enclosed in a poly bag to reduce transpiration. Phloem exudate was collected from an incision made in the bark of the stem 10 to 20 mm below the insertion of the leaf and analysed for ~4C-activity and sugar content. Sugars in the exudate were separated by thin layer chroma= tography. Radioactive areas, after location with a spark-chamber radiochromatogram scanner, were cut out and counted in a liquid scintillation counter. Sugars were quantitatively estimated by gasliquid chromatography of their methyl derivatives. The introduced 14C-labelled sugar (sucrose, glucose, fructose, galactose) was always recovered as sucrose in the exudate within a 20 min period. The pH of the perfusing solution was monitored.

Proton co-transport

Introduction

The loading of sugars into the sieve element is generally considered to be an active process operating at high flux rates. Energy-dependence is suggested by the promotion of loading from the free space with exogenously applied ATP and the sensitivity of loading to metabolic inhibitors, Kinetic studies also indicate an active carrier-mediated transport of sugars during phloem loading (see Geiger, 1975, and references therein). However, the nature of the carrier and the specific means of coupling it to the potential energy of ATP has not been resolved. Among the characteristics of phloem saps are the high pH (7.4-8.7) and high potassium content (< 100 mM) the significance of which has not been elucidated. Evidence is presented here that these features may reflect the sugar loading mechanism, a linked proton-potassium exchange pump at the plasma membrane of the sieve element creating a suitable proton gradient for sugar co-transport.

Results

When 50 mM K phosphate buffer was added to the perfusing sugar solution the sucrose concentration and the 14C-activity of the exudate both increased over a 2 h period. This increase was greater at pH 5 than at pH 8 (see Table). With 50 mM Na phosphate buffer a similar response was obtained, but the increase in both sucrose concentration and 14C-activity was much less, although again the level was greater

Table 1. The effect of potassium and sodium phosphate buffers on the loading of 14C-labelled glucose into Ricinus phloem at two pH levels Perfusing solution K phosphate buffer

Na phosphate buffer

5 pH

8 pH

5 pH

8 pH

laC-activity cpm ml 1 exudate

6770

1250

2620

720

% increase sucrose conc. exudate

29.6

13.5

20.6

2.3

298

F. Malek and D.A. Baker: Proton Co-transport of Sugars

20mM

8

a leaf resulted in [14C]sucrose appearing in the exudate within 20 min. When the hollow petiole was perfused with a bicarbonate buffer, pH 9.8, [14C]glucose and [tr were detected in the perfusing solution in a 1 : 1 ratio (Fig. 2).

KCI sucrose

7 c

"O--

~O

Discussion |

o_

5~M

6

CCCP

o

Q.

5

0

i

i

~

i

i

i

1

2

3

4

5

6

Time

(hours)

Fig. 1. pH changes in the solution perfusing hollow Ricinus petioles. o - - o Tris buffer, followed by the addition of KC1 and then sucrose, e - - e 2% sucrose, phosphate buffer followed by the addition of 5 gM CCCP

14(]

= o

pH9.8

12(]

2

o

lOO .E 80

o

60

"T E E

40

cL

Transport of sugars and amino acids coupled to a sodium gradient is well known in a number of animal cell systems (see Heinz, 1972) and in bacteria sugarH + co-transport has been found (West, 1970). In Chlorella the uptake of sugar is also postulated to involve coupled movement down a proton gradient (Komor, 1973). Co-transport with protons may therefore be a more fundamental process than co-transport with sodium. Uptake of sugars from the free space in Ricinus petioles has been shown here to be promoted by low pH and the presence of potassium, observations consistent with proton co-transport. The observed pH reduction stimulated by potassium and lessened by 2% sucrose is also consistent with the co-transport process. The uncoupler CCCP induced a pH rise, which may be interpreted as the inhibition of a proton efflux pump while inward co-transport of protons and sugars continued. Reversal of the proton gradient with a high pH in the perfusing solution caused an outward movement of sugars. It might be argued that such a high non-physiological pH was damaging the membrane, but the fact that monosaccharides rather than sucrose appeared in the perfusing solution suggests that membrane integrity was preserved. The above results indicate that the uptake of

20

60

90

120 Time

150

180

210

240

free

space

(apoplast)

element

sieve

(minutes)

Fig. 2. The effect of the pH of the solution perfusing hollow Ricinus petioles on the efflux of t4C-labelled sugars. Only [14C]glucose and [14C]frnctose were detected in the perfusing solution

Sug

. . . . i"

H+

~>

(~

u g aror

I i

at pH 5 than at pH 8. The pH of a weakly buffered (1 mM Tris) solution perfusing the petiole was monitored and a gradual decline of pH was observed. This pH drop was enhanced by the addition of 20 mM KC1 to the perfusing solution and lessened by the addition of sugars in the perfusing solution (Fig. 1). When the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) was added to a perfusing solution of 2% sucrose at a concentration of 5 gM the pH was observed to rise (Fig. 1). a4CO2 fed to

9 H+
K+ High Low

K+

t'~

po r~//

Low

H*

High

K+

Fig. 3. A scheme for a proton efflux/potassium influx pump at the plasma membrane of the sieve element with proton co-transport of sugars down the resultant proton gradient

F. Malek and D.A. Baker: Proton Co-transport of Sugars

sugars from the free space into the sieve elements of Ricinus is coupled with the inward movement of protons down a gradient of increasing pH. It is postulated that the gradient is maintained by a linked proton efflux/potassium influx pump (Fig. 3). The considerable ATPase activity reported at the surface of sieve elements (Gilder and Cronshaw, 1973) may be associated with these ion movements and would account for the observed stimulation of sugar loading by ATP (Geiger, 1975). The proposed model is consistent with the composition of Ricinus and other phloem saps (Hall and Baker, 1972) and with the inhibition and stimulation of the sugar loading process by various substances and conditions. Work is continuing to further characterise this sugar-H + co-transport system.

299

References Geiger, D.R. : Phloem loading. In: Encyclopedia of plant physiology, vol. 1 Zimmermann, M.H. Milburn, J.A. eds., pp. 395 431. Berlin-Heidelberg-New York: Springer 1975 Gilder, J., Cronshaw, J. : The distribution of adenosine triphosphatase activity in differentiating and mature phloem cells of Nicotiana tabacum and its relationshi p to phloem transport. J. Ultrastruct. Res. 44, 388404 (1973) Hail, S.M., Baker, D.A.: The chemical composition of Ricinus phloem exudate. Planta (Berl.) 106, 131-140 (1972) Heinz, E. : Na§ transport of organic solutes. Berlin-Heidelberg-New York: Springer 1972 Komor, E. : Proton-coupled hexose transport in Chlorella vulgaris. F.E.B.S. Letters 38, 16-18 (1973) West, I.C.: Lactose transport coupled to proton movements in Escherichia coll. Biochem. Biophys. Res. Commun. 41, 655 661 (1970) Received 2 March," accepted 2I March 1977

Proton co-transport of sugars in phloem loading.

Loading of (14)C-labelled sugars from the hollow petiole of Ricinus communis L. was stimulated by potassium and by low pH in that both the (14)C-activ...
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