Changing rates of uptake of [3H] leucine and other compounds during culture of tobacco mesophyll protoplasts

Plant Science Letters, 8 (1975) 197.-204 © Elsevier/North-Holland Scientific Publishers , Ltd.

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CHANGING RATES OF UPTAKE OF [3H]LEUCINE AND OTHER COMPOUNDS DURING CULTURE OF TOBACCO MESOPHYLL PROTOPLASTS

DAVID J. ROBINSON and MICHAEL A. MAYO Scottish Horticultural Research Institute, Invergowrie, Dundee (Scotland) (Received July 13th, 1976) (Revision received September 2nd, 1976) (Accepted September 10th, 1976)

SUMMARY

The rate of uptake of [3H]leucine by tobacco mesophyll protoplasts increased several-fold during the first 8 h in culture at 22°C, remained at this higher level for about 12 h, and then declined slowly until after 45 h in culture it was at or below the original rate. The increase in rate was observed not only with protoplasts in various physiological conditions but also with isolated cells, and was due neither to recovery from the trauma of centrifugation nor to depletion of a nutrient or accumulation of a product in the medium. It was prevented by cycloheximide or puromycin but not by chloramphenicol. Although the actual rates of uptake were different, similar changes occurred with [3H]uracil, [3H]UTP, [14C]giucose and [32P]phosphate.

INTRODUC~ON

Isolated protoplasts are useful in the study of plant biochemistry because, among other advantages, they have a greatly reduced diffusion path. We have used tobacco mesophyll protoplasts to study the effect of infection with tobacco rattle virus on metabolism. However, we found that the rate of uptake of various radioactively labelled precursors by the protoplasts did not remain constant during our experiments. Accordingly we have examined the changes in the rates of uptake of several radioactively labelled compounds during culture of the protoplasts. We also report some experiments designed to investigate the physiological and biochemical basis of these changes. METHODS

Preparation of protoplast cultures Nicotiana tabacum cv. Xanthi plants were grown as described by Kubo et al. Abbreviation: TCA, trichloroacetic acid.

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[ 1 ], and leaf mesophyll protoplasts prepared from them as described by Kubo et al. [2]. The protoplasts were resuspended in incubation medium [3], containing nystatin (E.R. Squibb & Sons, Liverpool; 25 units/ml) and carbenicillin (Beecham Research Laboratories, Brentford; 250/~g/ml), at a final concentration of 1.4--3.2- 10 s protoplasts/ml, and transferred to flat-bottomed tubes (5.3 × 1.7 cm) in 1 ml portions. Chloramphenicol (100 #g/ml), which did not affect uptake rates, was added in some experiments to inhibit further the growth of microorganisms. The tubes were kept in controlled environment cabinets at 22°C, and illuminated continuously at an intensity of 3000 lux. Inoculation with tobacco rattle virus was by the indirect method of Kubo et al. [2] using phosphate buffer. Preparations of isolated cells were obtained by omitting the treatment with cellulase.

Measurement of uptake and incorporation Times in culture were measured from the completion of the cell~lase digestion. Radioactively labelled compounds and other reagents were added in a small volume, usually 10 ~1, to 1 ml samples of protoplast suspensions and the tubes were kept for 2 h at 22°C. The contents were then diluted with 11 ml 0.7 M mannitol and the protoplasts were collected by centrifugation at 80 g for 90 s and washed with 10 ml mannitol. They were suspended in 0.7 ml 5% (w/v) TCA and kept overnight at 4°C. The TCA-insoluble material, collected by centrifugation, was washed once with 0.7 ml 5% TCA, and the combined supernatant fluids used to estimate the radioactivity taken up by the protoplasts. In each experiment, and in the absence of inhibitors of protein synthesis, radioactivity incorporated into TCA-insoluble material was an approximately constant fraction (less than 25%) of the total taken up, and was ignored in calculating uptake. For 3H- and 14C-labelled compounds, the TCAsoluble extract was mixed with 10 ml NE 260 scintillator (Nuclear Enterprises Ltd., Edinburgh) and counted in an Intertechnique SL30 liquid scintillation spectrometer. 32p was estimated by (~erenkov counting [4] without adding scintillator. Counting efficiencies were about 25% for 3H, 90% for 14C and 40% for 32p. Incorporation of [3H]leucine was measured by washing the TCAinsoluble material twice with ethanol and dissolving it in 0.5 ml Protosol (NEN Chemicals GmbH, Frankfurt). Samples were counted after adding 5 ml 0.5% PPO and 0.05% POPOP in toluene. All samples were counted for 10 min, or until at least l 0 s counts had been accumulated. Radiochemicals L-[4,5-3H]leucine (54 Ci/mmole), [5,6-3H]uracil (51 Ci/mmole), [5-3H]uridine triphosphate (1 Ci/mmole), D-[U-14C]glucose (3 mCi/mmole), D-[1-14C]mannitol (32.3 mCi/mmole) and [32P]phosphate (125 Ci/mg P) were all obtained from the Radiochemical Centre, Amersham.

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RESULTS

The quantities of six widely assorted radiochemicals taken up b y protoplast preparations in 2 h periods beginning either 1 h or 20 h after preparation of the cultures were measured (Table I). All except [~4C]mannitol were taken up at much greater rates at the later time; indeed the proportion of the supplied [~4C]mannitol which remained associated with the protoplasts was close to the proportion of the incubation medium expected to remain after the washing procedure. An average protoplast with a diameter in the range 20 to 40 pm has a volume of the order of 10 -8 ml. Thus 10 s protoplasts o c c u p y around 0.1% of the volume of a 1 ml culture. It can be seen (Table I) that the protoplasts, particularly at the later time, t o o k up more than this proportion of all the substances except mannitol, implying that they were concentrated inside the protoplasts. TABLE I UPTAKE OF VARIOUS RADIOACTIVELY LABELLED COMPOUNDS BY PROTOPLASTS Each culture contained 1.4--1.9.105 protoplasts in 1 ml incubation medium. The labelled compounds were supplied for 2 h, beginning after 1 h or 20 h in culture. Amount supplied (#Ci)

% taken up by 10 5 protoplasts 1--3 h (A)

20--22 h (B)

Ratio of counts B/A

1.0 1.0 1.0 0.1 36.4 0.25

5.3 1.1 0.26 0.30 0.14 0.05

27.2 9.5 2.8 7.3 1.1 0.06

5.2 8.6 10.8 24.3 7.9 1.2

1.0 50.0

5.1 0.04

11.9 ,0.34

2.3 8.5

Expt. A [3H]Leucine [3H]Uracil [3H]UTP [l'C]Glucose [ 32P]Phosphatea [ l'C]Mannitol

Expt. B [ 3H ] Leucine [ 3~P]Phosphate

aNo potassium phosphate in incubation medium.

The ratios between the rates of uptake at late and early times were quite different for the six compounds. In expt. B of Table I, [3H]leucine and [32p]. phosphate were added to the same cultures and the uptake of both was measured. The results show that the rates of uptake and the extent of the change of rate of uptake differed from one batch of protoplasts to another, and moreover were different for two c o m p o u n d s taken up by the same culture of protoplasts.

200 We chose to examine the uptake o f [3H]leucine in more detail. [3H]leucine was supplied for selected 2 h periods after isolation and the a m o u n t taken up was measured (Table II). The rate increased during the first 8 h in culture, and then remained high during the n e x t 12 h. Thereafter it declined slowly until after 45 h in culture it was at or below the initial rate. This decline was n o t apparently due to death of the protoplasts, because by 45 h the concentration of intact protoplasts was still at least 90% of the initial value. For convenience in subsequent experiments, we compared uptake during two 2-h periods, one beginning i h and the other 20 h after preparation of the cultures. TABLE II UPTAKE OF [ 3H]LEUCINE BY PROTOPLASTS AFTER VARIOUS PERIODS OF INCUBATION AT 22°C Each culture contained 2.1.10 s (expt. A) or 1.6.105 (expt. B) protoplasts in 1 ml incubation m e d i u m . 1 ~Ci [ 3H]leucine was added at the times indicated.

Time in culture (h) 1--3 4--6 7--9 10--12 13--15 16--18 19--21 22--24 44--46

Uptake (nCi/10 s protoplasts) Expt. A

Expt. B

56 105 200 112 178 182 159 I01 --

43 -----153 --

31

Increasing the concentration of leucine from 0.02/~M in the above experiments to 0.1 mM by adding unlabelled leucine to the incubation medium did n o t alter the rate o f uptake of label either early or late in the incubation (Table III). Thus over this range, the q u a n t i t y of leucine taken up was proportional to the concentration supplied. For measurement of uptake of [32p]. phosphate, the incubation medium was usually modified by omitting potassium phosphate. However, Table III shows t h a t although including this comp o n e n t depressed the uptake rate at both times by about 30%, the same change of rate between 1--3 h and 20--22 h after isolation t o o k place. We next considered the possibility t h a t the change in uptake behaviour had a trivial cause in the way in which we manipulated our preparations. Table IV shows t h a t when protoplasts were sedimented after 20 h in culture and resuspended in the same inc~ubatiQn medium, or in fresh incubation medium, the same high rate of uptake was observed as in a parallel culture not sedimented, implying t h a t the increased rate was due neither to recovery

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TABLE III UPTAKE O F [SH]LEUCINE OR [s2p]PHOSPHATE SUPPLIED AT D I F F E R E N T SPECIFIC ACTIVITIES 1 ~Ci [ SH]leucine and appropriate amounts of unlabelled L-leucine were added to culture~ containing 1 . 6 . 1 0 s protoplasts in 1 ml incubation medium. 50 ~Ci [32P]phosphate was added to cultures containing 2.3 • 10 s protoplasts in 1 ml incubation medium with, or 3 . 2 . 1 0 s protoplasts in 1 ml incubation medium without, potassium phosphate. The labelled compounds were supplied for 2 h, beginning after I h or 20 h in culture. [Leucine] uM

0.02 2.0 100

Uptake (nCi/10 s protoplasts) 1--3 h (A)

2 0 - 2 2 h (B)

20 27 22

168 186 193

Ratio B/A

8.4 6.9 8.8

[Phosphate] uM 0.014 2000

7.9 5.6

33.8 24.8

4.3 4.4

TABLE IV UPTAKE OF [3H]LEUCINE BY PROTOPLASTS AND CELLS; E F F E C T O F VARIOUS TREATMENTS Each culture contained 1 . 6 . 1 0 s protoplasts or 2 . 1 . 1 0 s cells in 1 nd incubation medium or 0.7 M mannitol. 1 uCi [3H]leucine was supplied for 2 h, beginning after 1 h or 20 h in culture. Treatment

Uptake (nCi/10 s protoplasts)

Ratio B/A

1--3 h.(A)

20--22 h (B)

Expt. A None R e s u s p e n d e d i n same medium at 20 h Resuspendedin ~ e s h m e d i u m a t 20 h

20 ---

168 198 148

8.4 9.9 7.4

Expt. B None Culture in 0.7 M-mannitol Cells

43 92 85

153 171 162

3.6 1.9 1.9

from the trauma of centrifugation nor to depletion of a nutrient or accumulation of a metabolic product in the medium. Although the incubation medium was poorly buffered, the pH changing from 5.8 to about 5.6 during culture, the addition of 2-(N-morpholino)-ethane sulphonate buffer pH 5.8 to 25 mM

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in the incubation medium did n o t affect the uptake rates although it did suppress the change in pH. When 0.7 M mannitol was used instead of the incubation medium, the rate of uptake of [3H]leucine still increased during incubation (Table IV, expt. B) although the extent of the change was smaller; furthermore, as with incubation medium (Table II), the rate of uptake 45 h after isolation had returned to its original level. Thus the change in uptake rate requires none of the other components of the incubation medium. Qualitatively similar changes in the rate of uptake of~3H]leucine were also observed in protoplasts that had been stored overnight at 4°C in 0.7 M mannitol before use [2] and in protoplasts inoculated with tobacco rattle virus. A similar though somewhat smaller effect was observed when isolated cells were cultured; the uptake rate both increased (Table IV) and subsequently declined to the initial level. Thus similar effects took place in protoplasts in a wide variety of physiological conditions and were at least not entirely due to removal of the cell wall. Inhibition of protein synthesis by cycloheximide (1 ~g/ml) or puromycin (0.3 mg/ml) added at the start of incubation prevented the increase in rate of uptake of [ 3H] leucine (Table V). Incorporation of [ 3H]leucine into acidinsoluble material was inhibited 82% by cycloheximide and 54% by puromycin, although when added together with [3H]leucine at either time, neither inhibitor affected the rate of uptake directly. However although cycloheximide at 0.3 ~g/ml inhibited incorporation by 58%, this concentration had little effect on the increase in uptake rate. Chloramphenicol (100 ~g/ml) did not alter the uptake behaviour of protoplasts.

TABLE V E F F E C T OF INHIBITORS ON UPTAKE OF [SH]LEUCINE BY PROTOPLASTS

Each culture contained 1.9. l 0 s (expt. A) or 1 . 8 . 1 0 s (expt. B) protoplasts in 1 ml incubation medium. 1 ~Ci [SH]leucine was supplied for 2 h, beginning after 1 h or 20 h in culture. Inhibitors were added after 1 h in culture at the concentrations shown..Inhibition of incorporation refers to cultures labelled after 20 h in culture. Inhibitor

Uptake (nCi/10 s protoplasts) 1--3 h (A)

Ratio B/A

Inhibition of incorporation

20--22 h (B)

(%)

ExpL A None C y c l o h e x i m i d e ( 1 #g/ml) P u r o m y c i n ( 0 . 3 mg/ml)

41 49 47

92 38 38

2.2 0.8 0.8

-82 54

52 55 61

216 218 74

4.2 4.0 1.2

-58 81

Expt. B None Cyc l o h ex i m i d e(0 . 3 ~g/ml) C y c l o h e x i m i d e ( 1 ug/ml)

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DISCUSSION

All the c o m p o u n d s that we have studied which were taken up to a significant extent were taken up b y protoplasts more rapidly after 20 h in culture than shortly after isolation, although the extent of the increase varied from one c o m p o u n d to another. All five c o m p o u n d s were concentrated in the protoplasts relative to the incubation medium; such transport against a concentration gradient presumably requires metabolic energy [5]. The increase in uptake rate was n o t prevented by chloramphenicol, which inhibits protein synthesis on chloroplast ribosomes, but was prevente-~ both by cycloheximide, which inhibits protein synthesis on cytoplasmic riuosomes, and by puromycin, which inhibits both [6]. If, as seems likely [5], the active transport of the c o m p o u n d s we have studied is mediated by substrate-specific carrier proteins then the increases in transport rates apparently stem from synthesis of additional carriers. However, substances as varied as those we have used are probably transported by different carriers. It is n o t clear why there should be a general stimulation of carrier synthesis during culture of the protoplasts. Our experiments suggest that it is n o t a consequence of recovery from damage caused either by the removal of the cell wall or by subsequent centrifugation. Nor does it seem to result from depletion of a c o m p o n e n t of the incubation medium or accumulation of an excreted product. In fact it occurs with protoplasts in many different conditions, although its extent may vary, as indeed it does between one batch of protoplasts and another. However some procedures, such as exposure to high osmotic pressure, are unavoidable during protoplast preparation, and it may be that the increase of uptake rate reflects recovery from such a procedure. Recent experiments [7] have shown that the rate of uptake of phosphate ions by maize protoplasts increases during the first few hours in culture, and that protoplast isolation procedures can alter the rates of efflux of ions from cells. Changes in rates of efflux during culture may explain the apparent decline in uptake we have observed at later times. When slices are prepared from a variety of plant tissues, they develop an increasing capacity for solute uptake, which has been described as the "washing" or "ageing" phenomenon. This effect, which has been interpreted in terms of derepression [8], might be related to the one we have observed in protoplasts. Whatever the mechanism involved, ~the practical implication of this work is clear. In studies of the incorporation of tracers into macromolecules in protoplasts, it is important to measure the uptake of the tracer and to relate incorporation to uptake. Indeed the rates of uptake of some c o m p o u n d s at some stages of incubation are so r a p i d t h a t even over as short a period as 2 h, the extracellular concentration may fall significantly, making kinetic experiments very difficult to interpret. In addition it should b e borne in mind that although those c o m p o u n d s with which we have observed changing uptake rates are normal cellular metabolites, other substances may behave in a similar way. For

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example, application of the same extracellular concentration of a drug or inhibitor at different times m a y give rise to quite different intracellular concentrations. ACKNOWLEDGEMENT We t h a n k L i n d a Cable f o r t e c h n i c a l assistance.

REFERENCES 1 2 3 4 5 6 7 8

S. Kubo, B.D. I-Iarrimonand H. Barker, J. Gen. Virol., 28 (1975) 255. S. Kubo, B.D. Harrison, D.J. Robinson and M.A. Mayo, J. Gen. Virol., 27 (1975) 293. Y. Otsuki, T. Shimomura and I. Takebe, Virology, 50 (1972) 45. J.M. Gould, R. Cather and G.D. Winget, Anal. Biochem., 50 (1972) 540. E. Epstein, Int. Rev. Cytol., 34 (1973) 123. D. Boulter, Annu. Rev. Plant Physiol., 21 (1970) 91. A.R.D. Taylor and J.L. Hall, J. Exp. Bot., 27 (1976) 383. R.F.M. van Steveninck, Annu. Rev. Plant Physiol., 26 (1975) 237.