Plant Science Letters, 12 (1978) 93--99 93 © Elsevier/North-Holland Scientific Publishers Ltd.

EVIDENCE FOR AN ACTIVE TRANSPORT OF METHYL-a-D-GLUCO- PYRANOSIDE IN PEA STEM SEGMENTS

MARIA IDA DE MICHELIS*, MARCO RADICE**, ROBERTA COLOMBO* and PIERA LADO*.

*Centro di Studio del C.N.R. per la Biologia Cellulare e Molecolare delle Piante, Istituto di Scienze Botaniche, Universitd di Milano and **Montedison S.p.A., Ricerca e Sviluppo, Instituto G. Donegani, Novara (Italy)

(Received November 3rd, 1977) (Accepted January 30th, 1978)

SUMMARY

The nature of the transport of ~-MG in pea internode segments has been investigated, a-MG uptake exhibits saturation kinetics. The transport of a-MG occurs against a concentration gradient and is inhibited by inhibitors of oxida- tive phosphorylation (FCCP and Antimycin A), indicating that an active pro- cess is involved in ~-MG transport. This conclusion is in agreement with the finding that a-MG transport is strongly enhanced by fusicoccin which has been shown to stimulate several active transport processes in plant materials.

INTRODUCTION

A large evidence indicates that methyl~-D-glucopyranoside (a-MG) is actively transported in bacteria [1--3], yeasts [4--6] and in animal tissues (hamster intestine and rat kidney slices). In animal tissues a-MG is probably taken up by the same carrier involved in glucose and galactose active transport [7,8], while in microorganisms there is some evidence that a-MG is transported by the glucose or maltose uptake systems [1,2,5].

No detailed investigation aimed to characterize a-MG transport in algae and higher plants has been reported. However in castor bean cotyledons this sugar has been used as a substance entering the cell by passive permeation [9,10]. In the present work we have studied a-MG uptake in pea internode segments and we present evidence for active a-MG uptake occurring in this material.

Abbreviations: ~-MG, methyl~-D-glucopyranoside; WFS, water free space; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; FC, fusicoccin.

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MATERIALS AND METHODS

Pea (Pisum sativum L. cv Progress 9) internode segments (5 mm long) were cut from the subapical part of the distal internode of the seedlings, grown on poplar sawdust for 6 days at 25°C in the dark. The segments were harvested and incubated for 45 min in 5 × 10 -4 M CaC12 + 2.5 × 10 -4 MgC12, then rapidly washed and transferred to the various media as specified in the single experiments. The incubation was always performed in 5 X 10 -3 M K-phosphate buffer at pH 5.5 in the presence of 5 X 10 -4 M CaC12 and 2.5 X 10 -4 M MgC12. The tissue volume ratio was approx. 40 mg/ml in all the experiments. The experiments were performed in a water thermoregulated bath with shaking (70 spm) at 25°C in the dark.

All the experiments were performed with 3 replicates. Data are averages from two or more experiments.

Measurements of a-MG uptake The samples were incubated in the presence of a-methyl-D-gluco[U-14C]-

pyranoside (The Radiochemical Centre, Amersham) at the concentration and for the time specified in the single experiments.

At the end of the incubation the segments were washed as specified in the single experiments. Then the tissue was homogenized in 4 ml of water and resuspended in 10 ml of Insta-gel (Packard). The radioactivity of the samples was counted with a Packard Tricarb Scintillator. Standard deviation for uptake values does not exceed + 4%.

Measurements of a-MG efflux The samples were incubated for 4 h in the presence of 10 -3 M labelled a-MG,

rapidly rinsed and transferred in an a-MG-free medium. At the desired times an aliquot of the medium was withdrawn for assay of radioactivity and the incuba- tion medium was replaced. At the end of the treatment the radioactivity of the samples was assayed as described above.

Lack of metabolism of a-MG within the tissue The following experiments indicate that no appreciable a-MG metabolism

occurs within the tissue during uptake measurements. The amount of radio- activity present in CO2 evolved during the uptake of labelled a-MG was measur- ed. Respiratory CO2 was trapped with hydroxide of hyamine, which was assay- ed for radioactivity. The radioactivity of CO2 accounted for less than 4% of a-MG taken up. Chromatographic analysis of an extract of the tissue was per- formed to check the distribution of the labelling of 10 -a M a-MG taken up by the tissue during 24 h of incubation. The tissue was extracted with 10% trich- loroacetic acid or with ethanol at 70°C. In both cases more than 90% of the radioactivity was found in the soluble fraction. The ethanolic extract, concen- trated ip a vacuum desiccator, was chromatographed on thin layer cellulose in butanol--ethanol--H20 (52 : 33 : 15). All the radioactivity, revealed by auto- radiography was found in a single spot with Rf corresponding to that of a-MG.

95

R E S U L T S

Kinetics o f a-MG uptake as a function o f external concentration Table I shows the values of a-MG uptake (measured over a 2-h period) in the

range of external a-MG concentration between 10 -4 and 10 -1 M. The ratio between the rate of a-MG uptake and its concentration in the medium consider- ably decreases (from 0.14 to 0.057) as the external concentration increases, indicating a saturation kinetics. This behaviour favours the view that a-MG up- take depends on a carrier-mediated process rather than on simple physical diffusion. The fact that the rate of a-MG uptake is still increasing also at the highest concentrations tested may be explained either by very low affinity of the carrier for a-MG or by superimposition of simple physical diffusion to the carrier-mediated uptake. Saturation kinetics shown in our material apparently contrast with the kinetics of simple diffusion reported by Komor et al. [9] for a-MG transport in castor bean cotyledons. This difference may suggest that in castor bean cotyledons and in pea stems a-MG is taken up by different pro- cesses. There is evidence that in some plant materials sugars can be taken up by a saturable system simultaneously with simple diffusion [9,13--15]. If this is true also in cotyledons and in stems, a-MG could be taken up mainly by diffus- ive process in the former and by carrier-mediated system in the latter. Alterna- tively, in cotyledons the carrier for a-MG transport might have such a low affinity that uptake kinetics is practically linear in the concentration range tested.

T A B L E I

RELATIONSHIP BETWEEN THE RATE OF a-MG UPTAKE AND ITS CONCENTRA- TION IN THE EXTERNAL MEDIUM.

T h e samples were i n c u b a t e d in t h e p re sence o f labe l led a -MG at the specified c o n c e n t r a - t ions fo r 2 h. A t t h e e n d o f t he i n c u b a t i o n t h e t i s sue was r ap id ly r insed , t h e n w a s h e d for 10 m i n in ice co ld un l abe l l ed s o l u t i o n w i t h shak ing a n d r insed again.

a - M G in t h e a - M G uptake m e d i u m (~ m o l • g- 1 ( raM) ft. wt . • 2 h -~ )

T C F a

0.1 0 . 0 1 4 0 . 1 4 0 0.3 0 .048 0 . 1 6 0 1.0 0 . 1 5 3 0 . 1 5 3 3.0 0 . 4 1 2 0 .137

10.0 1 .043 0 . 1 0 4 30 .0 2 .391 0 . 0 8 0 60 .0 3 . 8 8 0 0 . 0 6 5

100 .0 5 .665 0 .057

a T F C ( t i ssue c o n c e n t r a t i o n f ac t o r ) r e p r e s e n t s t h e r a t io : ~ m o l ~-MG • g-~ fr. wt . in the t i s s u e / u m o l ~ -MG • ml -~ in t he m e d i u m .

96

i ° 4 m

x 3 - - O E

-~ 2 ¢o

L9 jo/

I 8

~-MG concen t ra t ion

in the med ium (/~mol • ml-I )

0.05 0.20 1.00

50.00

in the t issue (p.mol og-i fr. wt.)

0 .22 0.85 4.73

91 .40

| I 16 24

Fig. 1. A c c u m u l a t i o n o f ~-MG w i t h i n t h e t issue. T h e graph shows t h e u p t a k e o f 10 -3 M ~- MG as a f u n c t i o n o f i n c u b a t i o n t ime . T h e da t a in t h e inse r t r ep re sen t ~-MG u p t a k e a f t e r 24 h o f i n c u b a t i o n in t h e p re sence o f var ious a - M G c o n c e n t r a t i o n s . At t he e n d of the incuba- t i o n t he s egmen t s were r insed t h r ee t i m e s w i th 5 ml o f dis t i l led water .

Accumulation o f a-MG against concentration gradient A second point investigated was whether ~-MG uptake is an active or a pass-

ive process. The data of Fig. 1 show that ~-MG can be taken up against concen- tration gradient. ~-MG is not appreciably metabolized in this material (see Materials and Methods), thus the radioactivity values represent an effective measure of the concentrat ion o f ~-MG within the tissue.

The data show tha t the uptake of 10 -3 M ~-MG lasts at an almost steady rate at least up to 24 h, in spite of the fact tha t ~-MG concentrat ion within the tissue equals external concentrat ion already after 5--6 h. After 24 h the concen- t rat ion o f ~-MG within the tissue reaches values more than 4 times higher than the external one. Accumulation of ~-MG occurs in the whole concentrat ion range tested (insert of Fig. 1).

Release o f a-MG taken up The segments were preloaded with a-MG by incubating the tissue for 4 h in

10 -3 M ~-MG, then transferred to an ~-MG-free medium. The kinetics of a-MG release from the tissue in these conditions is shown in Fig. 2. The efflux rate, initially very rapid, sharply decreases to very low values in the following period. Most of the amount of a-MG released in the first 15 min (0.16 pmol/g fr. wt., corresponding to 19% of total ~-MG uptake) is conveniently interpreted as due to diffusion from the WFS as it corresponds to the ~-MG contained in the WFS in equilibrium with 10 -3 M external ~-MG (WFS in this material is estimated as accounting for 15--20% of total tissue volume [11] ).

i

0.10

X

40,15

0.2

97

0

5

¢0

l O ~-

0

15 ~- 0

20

25

"/ 0

0.05

J 2 ~

I ! | = ~ ! 0 1 2 3 ~ 24

HOURS

Fig. 2. Release o f a - M G t a k e n up. T h e release o f a -MG f r o m pea i n t e r n o d e s egmen t s pre- loaded w i th 10-3 M label led a - M G fo r 4 h , was m e a s u r e d as desc r ibed in t he Mater ia ls and Methods . a -MG release is expressed as u m o t * g-~ fr. wt . ( lef t scale) or as % of t o t a l a -MG u p t a k e ( r ight scale).

Very little a-MG is released after 15 min and after 24 h more than 70% of total a-MG taken up is still retained within the tissue. In contrast a-MG is com- pletely released when the tissue is frozen-thawed four times (data not shown) ruling out the possibility that a-MG retention depends on aspecific absorption on the tissue.

These data taken as a whole indicate that passive diffusion must play only a minor role in a-MG transport in our material.

Inhibition of a-MG uptake by FCCP, Antimycin A and low temperature The data of Table II show that at least a considerable fraction of a-MG up-

take depends on energy availability, as expected for an active transport process. In fact FCCP and Antimycin A, inhibitors of oxidative phosphorylation, lower the rate of a-MG uptake by approx. 56% or 43% respectively over a 2-h period.

The uptake of a-MG is drastically (by approx. 80%) inhibited by lowering the temperature from 25°C to 5°C (data not shown). This finding is consistent with the view that a-MG uptake depends on cell metabolism. A passive trans- port cannot however be ruled out, owing to the well known dependence of the fluidity of membrane lipids on temperature changes.

Effect of FC on a-MG uptake FC has been shown to stimulate in many plant materials active uptake of

cations, inorganic and organic anions, aminoacids and sugars [12,13], whilst it does not affect water permeability and the transport of the passively permeat-

9 8

TABLE II

EFFECT OF 10 -4 M FCCP, 10 -5 M ANTIMYCIN A, 2 × 10 -s M FC ON a-MG UPTAKE

The samples were incubated for 2 h in the presence of labelled a-MG at the specified con- centrat ions . A t the end o f i n c u b a t i o n the t i ssue was rapidly rinsed, then washed for 10 rain in ice co ld unlabe l led s o l u t i o n with shaking and rinsed again. Data are expressed as p m o l - g-~ ft. wt. • 2 h -1 . The figures in the brackets represent the effects of the various treat- ments, as percent of the controls.

aoMG Control FCCP A n t i m y c i n A FC

10 -4 M 0.014 0.006 (-56%) -- 10 -3 M 0.153 0.066 (-57%) 0.087 (-43%) 0.362 (+137%) 10 -2 M 1.043 -- -- 2.630 (+152%)

ing subs tances u rea and t h iou rea [ 1 6 ] . We have thus invest igated w h e t h e r FC af fec t s the u p t a k e o f a-MG. The da t a o f Table 2 show th a t FC m a r k e d l y {approx. 150%) s t imula tes a -MG up take .

CONCLUSIONS

The da t a r e p o r t e d above p resen t ev idence fo r a car r ie r -media ted t r an sp o r t o f a -MG in pea i n t e m o d e segments . This t r an sp o r t appears t o be active, as it occurs against a c o n c e n t r a t i o n gradient , and it is un id i rec t iona l and energy- d e p e n d e n t . This conc lus ion is in ag reemen t wi th the f inding tha t a-MG up take is s t rongly s t imula ted b y FC, which has been shown to s t imula te active trans- p o r t sys tems.

The fac t t ha t in the pea i n t e m o d e segments a-MG is act ively t r anspo r t ed suggests t ha t careful con t ro l s are necessary be fo re using it as a passive p e r m e a n t in h igher p lan t materials .

ACKNOWLEDGEMENTS

The p resen t s t udy is pa r t o f a coopera t ive research p rogram on t r anspo r t sys tem in plants , wi th the par t i c ipa t ion o f Cen t ro di S tud io del C.N.R. per la Biologia Cellulare e Molecolare delle Piante , I s t i tu to di Scienze Botan iche , Universi t~ di Milano, Milan ( I ta ly) and o f the I s t i tu to Donegani (Monted i son S.p.A.) , Novara (I ta ly) .

We t h a n k Prof . E. Marr~ fo r he lpfu l discussion and suggestions. Thanks are due to Agrochemica l Division o f Mon ted i son S.p.A., Milan {Italy) fo r the gift o f fusicoccin.

99

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