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Cultured carrot cell mutants: 5-methyltryptophan-resistance trait carried from cell to plant and back
Plant Science Letters, 3 (1974) 323--330 © Elsevier Scientific Publishing Company, A m s t e r d a m -- Printed in The Netherlands
CULTURED CARROT CELL MUTANTS: 5-METHYLTRYPTOPHANRESISTANCE TRAIT CARRIED FROM CELL TO PLANT AND BACK
J.M. WIDHOLM
Department of Agronomy, University of Illinois, Urbana, Ill. 61801 (U.S.A.) (Received March 1st, 1974) (Revision received June 24th, 1974)
SUMMARY
A mutant Daucus carota (carrot) cell line resistant to growth inhibition by 5-methyltryptophan was selected from normal cells by growth in the presence of this analog. Plants were regenerated from both normal and resistant cell cultures. Cell cultures were reinitiated from petioles of 32 plants which had originated from the resistant cell line and 4 from the normal line. All but one of these cultures derived from the resistant mutant, and none of those cultures derived from the normal plants, were resistant to growth inhibition by 5-methyltD, ptophan. The resistance to growth inhibition was apparently due to decreased uptake of the tryptophan analog and not due to altered feedback control of anthranilate synthetase as reported for other 5-methyltryptophan-resistant mutants described previously. INTRODUCTION
Increasing interest is being focused on the application of cell culture techniques to plant breeding. One useful application would involve the selection of desirable mutants from cell cultures which could then be used to regenerate plants possessing the desired trait. A question important to the utility of this method is whether mutations selected at the biochemical level in cultured cells can be carried to the regenerated plant as a stable trait. Two reports have appeared recently which show that mutations in two tobacco cell lines are stable. Carlson [1 ] selected cells resistant to methionine sulfoximine and grew plr,nts from these cells. Several of these plants exhibited increased free methionine levels and these plants gave some progeny resistant to the methionine analog. Maliga et al. [2] reported the selection of haploid tobacco callus in which growth was resistant to inhibition by streptomycin. Diploid plants were grown from this tissue and the callus formed from leaf sections also showed streptomycin resistance. Likewise, progeny grown from seeds produced by these plants were resistant. 323
Previous repoas from this laboratory [ 3,4] described tobacco and carrot cell mutants resistant to growth inhibition by DL- 5-methyltryptophan, a tryptophan analog. This resistance was due to the presence of an altered anthranilate synthetase which was more resistant to feedback inhibition by the analog, or tryptophan the natural inhibitor. These mutants were obtained from cell lines which had been cultured for many years and had lost the ability to regenerate complete plants. This report describes the selection of 5-methyltryptophan-resistant mutants from freshly initiated carrot cell lines and also describes the passage of this trait from cultured cells to plants and back into cells in culture. Studies concerning the mechanism of resistance are also reported, since the mutants describe~l here were different from those described previously [ 3,4] and did not have a resistant control enzyme. MATERIALS
AND METHODS
Garden carrot root cells (Daucus carota L.) were cultured and the mutant selected as described before [ 3,4] in liquid medium [5]. The DL-forms of all tryptophan analogs were used in the studies. Growth studies were initiated by inoculating 0.5 g fresh weight of suspended cells into 100 ml fresh medium containing the inhibitors at the concentrations listed. After 10 days incubation the cells were collected by vacuum filtration on Mirracloth and weighed. Plants were regenerated by transferring 0.5 ml suspended cells onto 50 ml of the medium described before [5] (lacking 2,4-D, but containing 1 mg/l each of indoleacetic acid and kinetin and 1% agar). Flasks were placed under continuous fluorescent light and after one month small aggregates of greening cells were transferred to fresh medium where complete plants were formed. These plants were placed in soil in pots and grown in the greenhouse. When the plants were about 20 cm tall, 2 cm segments of 1 petiole from each plant were sterilized with 1% sodium hypochlorite for 5 min and placed on regular carrot medium solidified with 1% agar. The callus which formed was used to initiate suspension cultures which were employed for the studies. Anthranilate synthetase activities and free tryptophan were measured as described previously [ 4]. The uptake of filter-sterilized DL- 5-methyltryptophan ( [ 14C ]methyl, 48.2 uCi/umole, CEA, France), DL-5-hydroxytryptophan ([ 3-14C]alanine, 4--7 pCi/umole, Calatomic), DL-tryptophan (2-ringJ4C, 26.7 pCi/pmole, Calatomic) and L-[ULJ4C]leucine (282 ~Ci//~mole, Mallinckrodt) was measured by incubating the cells (which were in the rapid growth phase) in fresh medium containing the labeled compounds at 27 ° . Sterile techniques and solutions were used throughout. At intervals, aliquots of the cell suspensions were collected on filter paper discs by vacuum filtration and rinsed 3 times with 2 ml water containing 0.1 mM of ~ach unlabeled compound. The methods of rinsing and radioabtivity determination were as described by Ferrari and Widholm [6]. At the end of each experiment an aliquot of the incubation medium was plated on nutrient agar plates to detect microbial 324
contamination, but no significant growth was found in the experiments reported here. RESULTS
Cells from a newly initiated garden carrot root culture were ir,oculated into liquid medium containing 220 vM DL-5-methyltryptophan. This concentration completely inhibited growth (increase in fresh weight) when measured after 14 days, but some flasks showed growth within 30 to 60 days. A resistant cell line was established by reinoculating the growing cells into fresh medium containing D L - 5 - m e t h y l t r y p t o p h a n where growth continued. Then, after several subcultures on selective medium, plants were regenerated from both the resistant and normal lines. Cell cultures were re-established from petioles obtained from these regenerated plants. Growth studies with the cultures started from regenerated plants showed that 31 of the 32 cell lines had remained resistant to growth inhibition by 5-methyltryptophan. Of the 4 lines started from plants regenerated from normal cells all were susceptible to growth inhibition. Further studies were carried out with 5 representative cell lines, the origins of which are diagrammed in Fig. 1. Microbial designations are used to describe the mutant lines. All the lines were derived from the original normal line, trp*. This line and the trpP-1 resistant line, which was selected from the trp ÷ line during growth on 5-methyltryptophan, had been in suspension culture continuously for 2 years. Also included was a representative trp ÷ and trpP line obtained from plants {trp ÷2 and trpP-2, respectively) which were grown from the trp ÷ and trpP-1 lines, respectively. Another cell line which had also been selected as resistant to growth inhibition by 5-methyltryptophan was also included. This latter cell line, as will be shown later, differs in the apparent mechanism of resistance and will be denoted trpE-1. The growth curves shown in Figs. 2 and 3 show that the resistant cells, Cell Cultures
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Fig.2. The effect of DL-5-methy|tryptophan on the growth of trp* (~'), trpP-1 (o), trp÷2 (-), lrpP-2 (A) and trpE-1 (~) cultured carrot cells. The study was carried out as described in MATERIALS AND METHODS with the final control weights being 11.7, 12.0, 11.0, 11.6 and 15.3 g, respectively. Fig.3. The effect of Db-5-hydroxytryptophan on the growth of trp* (c), trpP-1 (e), trp÷2 (::), trpP-2 (A) and trpE-I (~) cultured carrot cells. The experimental procedure and the weights of the control cultures were the same as in Fig.2.
whether trpP-1, trpP-2 or trpE-1, are much more resistant to growth inhibition by 5-methyltryptophan or 5-hydroxytryptophan, another tryptophan analog. The 5-hydroxy-analog of tryptophan was somewhat less effective in inhibiting growth of the trp+ cells. The resistant cells were also more resistant to growth inhibition by 5-fluorotryptophan and 6-fluorotryptophan (data not shown) the only other analogs tested. The previously described 5-methyltryptophan-resistant carrot and tobacco cell mutants [ 3,4] possessed anthranilate synthetase activity more resistant to inhibition by 5-methyltryptophan or by tryptophan than was the enzyme activity from the normal cells. Thus, the inhibition of the anthranilate synthetase activities in crude extracts obtained from the carrot cell lines was measured (Figs. 4,5,6) to determine if the same mechanism was operative. It is apparent that the enzyme found in both the trpP-1 and trpP-2 cells was not more resistant to inhibition by tryptophan, 5-methyltryptophan or 5-hydroxytryptophan, unlike the carrot and tobacco mutants described previously [ 3,4]. The cell line designated trpE-1 did have a less sensitive enzyme, but plants have not as yet been grown from this line. None of the 31 trpP-2 cell lines was found to have a feedback insensitive enzyme. The free tryptophan content of these lines was also determined since high levels had been previously found in cells containing the le~s sensitive enzyme [3,4]. In the present study the trp ÷and trp÷2 cells had low levels, 70 326
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Fig.4. Effect o f L-tryptophan on anthranilate synthetase activity in crude extracts from trp ÷ (o), trpP-1 (e), trp ÷2 (~), trpP-2 (A)and trpE-1 (o) cultured carrot cells. Each assay contained 0.2 ml enzyme extract containing 1.38, 1.08, 1.08, 0.88 and 0.76 mg protein and the contrc.ls produced 7.5, 7.5, 11.4, 10.0 and 9.0 nn'loles anthranilate in 30 rain, respectively. Fig.5. Effect of DL-5-methyltryptophan on anthranilate synthetase activity in crude extracts from lrp ÷ (c), trpP-1 (e), trp ÷2 (~), trpP-2 (A)and trpE-1 (u) cultured carrot cells. The extracts and control activities were the same as Fig.4. Fig.6. Effect of DL-5-hydroxytryptophan on anthranilate synthetase activity in crude extracts from trp ÷ (o), trpP-1 (e), trp .2 (:,), trpP-2 (A) and trpE-1 (u) cultured carrot cells. The extracts and control activities were the same as Fig.4.
and 197 nmoles tryptophan/g fresh weight, respectively. The trpP.1 and trpP.2 cells contained 644 and 906 nmoles/g, respectively, while the trpE.1 cells contained 2800. The resistant cells contained significantly higher free tryptophan than the + cells, and the line with the altered enzyme trpE.1 over 3-fold more than the other resistant cells. The 4 other plant trpP-2 lines examined contained from 772 to 962 nmoles/g fresh weight of free tryptophan. Since penetration of the inhibitor into the cells could confer resistance, measurements of the rate of uptake of DL-[ 14 C]-5-methyltryptophan and D L - [ ~ 4 C ] - 5 - h y d r o x y t r y p t o p h a n were made. Both analogs were used since 5-hydroxytryptophan apparently acts in a manner identical to that of 5methyltryptophan [ 5 ]. Fig. 7 shows the uptake of these two analogs and DL-[14C]tryptophan and L-[~4C]leucine by trp ÷ and trpP-1 cells. The uptake of DL- [~4C]-5-methyltryptophan by the trpP-1 cells was less than one-third that by the trp ÷ cells. The uptake of DL-[ 14 C]-5-hydroxytryptophan or DL- [14C]tryptophan by the trpP-1 cells was less than half that of the bp ÷ cells during the 3-h uptake period. The uptake of [~4C] leucine was identical in both cell types. The cells used in these experiments were growing at identical rates in early log phase of growth. Table I lists the results of an experiment comparing the rates of uptake of 327
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INCUBATION TIME (h) Fig.7. Uptake of ~'C-labeled DL-5- methyltryptophan (SMT), DL-5 -hydroxytryptophan (SHT). D~tryptophan (TRP) and L-leucine (LEU) by trp + (~) and trpP-1 (e) cells. Early exponential growth phase cells (125 mg) were incubated at 27 ° in 25 ml fresh medium containing 1 . C i of each labeled compound. Duplicate I ml samples were removed at time zero and at the times shown from each of 2 duplicate flasks and rinsed with 3 × 2 ml water washes containing 10 -4 M of each unlabeled compound on filter paper discs in a suction flask as described by Ferrari and Widholm [6]. The ~4C in the washed ceils on the filters was determined in a scintillation spectrometer and the standard deviations are denoted by the bars.
TABLE I UPTAKE OF DL- [ '" C ]-5-HYDROXYTRYPTOPHAN BY CULTURED CARROT CELLS Cells (100 mg fresh weight) wert pipetted into 10 ml fresh medium containing 0.1 , C i DL-[ z4C ]-5-hydroxytryptophan and at the times shown, 1-ml aliquots of the cell suspension were removed to determine the uptake as described in MATERIALS AND METHODS. Each point represents one determination. Cell line
t rp + trpP-1 trp÷" trpP-2 trpE-1
328
Uptake of DL- [' 4C ]-5-hydroxytryptophan (cpm/10 mg cells) 3h
6h
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2212 496 2760 931 802
DL- ['4C]-5-hydroxytryptophan by the 5 cell lines being studied (trp ÷, trp+2, trpP-1, trpP-2 and trpE-1). The resistant lines took up much less of the analog at the 3- and 6- h periods than did the + lines. This reduced uptake of 5hydroxytryptophan by the resistant cells was not due to a lower growth rate as no differences in growth rates of the 5 cell lines were noted when grown in a medium without inhibitor. DISCUSSION
The information presented shows that the 5-methyltryptophan-resistance trait can be carried from carrot cell cultures to plants and back into culture. This indicates that this trait is quite stable. No studies on the plants were carried out, except to measure the free tryptophan in the leaves and roots. No reproducible differences in tryptophan levels were noted in plants of trp ÷ or trpP-1 origin. The mechanism of resistance observed with the carrot mutants studied here is, however, apparently different from that observed with both carrot and tobacco mutants described previously [ 3,4 ]. In the tobacco and carrot mutants described previously [ 3,4], the control enzyme, anthranilate synthetase, was less sensitive to feedback inhibition by 5-methyltryptophan or t r y p t o p h a n the natural inhibitor. This resistant enzyme could explain the resistance of these cell lines to growth inhibition by 5-methyltryptophan and also why they accumulate 20 to 30 times the normal free tryptophan. The evidence indicates that the uptake of 5-methyltryptophan by the trpP cell lines, described in this report, is less than one-third that of the trp ~ cells (Fig. 7). The trpP cell lines apparently take up less inhibitor and may in this way be able to grow in the presence of the analogs. Since the cells used were apparently diploid at the time of selection, a permeability mutant may not be expected to be found since both genes coding for the proteins controlling uptake might have to be altered. However, since some of the analog is taken up, the uptake results might be consistent with a heterozygous mutation. Despite the presence of the apparently normal control enzyme, the trpP cells studies here had increased levels of free tryptophan, though not as high as in the cells which had a 5-methyltryptophan resistant anthranilate synthetase (trpE-1). Why the free tryptophan is increased in these trpP cells is not known. This might be explained by a change in membrane permeability which keeps the cellular tryptophan more securely within the cell or more effectively compartmentalized away from the control enzyme. There is evidence indicating that tryptophan is extensively compartmentalized in cultured cells [7 ]. The trpP-1 cells take up less than one-third as much 5-methyltryptophan (Fig. 7), yet require about 30 times as much analog in the medium for equal growth inhibition when compared to trp ÷ cells (Fig. 2). The reason for this difference between uptake and medium inhibitory levels is not known, but more effective compartmentation might play a role.
329
Whether t h e D or L forms of 5 - m e t h y l t r y p t o p h a n are t a k e n up and act as ~ inhibitors is likewise n o t known. The L, b u t n o t the V form of t r y p t o p h a n is an effec¢ive inhibitor of anthranilate synthetase from carrot cells, however [ 4]. Tobacco cells can take u p D - t r y p t o p h a n and racemize it enzymatically to L-tryptophan [8 ]. ACKNOWLEDGEMENTS
This research was supported in part b y t h e Illinois Agricultural Experiment Station and was done with t h e technical assistance of Rodger Shewmaker. REFERENCES 1 2 3 4 5 6 7 8
P.S. Carlson, S~ence, 180 (1973) 1366. P. Maliga, A. Sz.-Breznovits and L. Marton, Nature New Biol., 244 (1973) 29. J.M. Widholm, Biochim. Biophys. Acta, 261 (1972) 52. J.M. Widholm, Biochim. Biophys. Acta, 279 (1972)48. J.M. Widholm, Biochim. Biophyso Aeta, 261 (1972) 44. T.E. Ferrari and J.M. Widholm, Anal. Biochem., 56 (1973) 346. J.M. Widholm, Physiol. Plant., 30 (1974) 323. G.A. Miura and S.E. Mills, Plant Physiol., 47 (1971) 483.
330
CULTURED CARROT CELL MUTANTS: 5-METHYLTRYPTOPHANRESISTANCE TRAIT CARRIED FROM CELL TO PLANT AND BACK
J.M. WIDHOLM
Department of Agronomy, University of Illinois, Urbana, Ill. 61801 (U.S.A.) (Received March 1st, 1974) (Revision received June 24th, 1974)
SUMMARY
A mutant Daucus carota (carrot) cell line resistant to growth inhibition by 5-methyltryptophan was selected from normal cells by growth in the presence of this analog. Plants were regenerated from both normal and resistant cell cultures. Cell cultures were reinitiated from petioles of 32 plants which had originated from the resistant cell line and 4 from the normal line. All but one of these cultures derived from the resistant mutant, and none of those cultures derived from the normal plants, were resistant to growth inhibition by 5-methyltD, ptophan. The resistance to growth inhibition was apparently due to decreased uptake of the tryptophan analog and not due to altered feedback control of anthranilate synthetase as reported for other 5-methyltryptophan-resistant mutants described previously. INTRODUCTION
Increasing interest is being focused on the application of cell culture techniques to plant breeding. One useful application would involve the selection of desirable mutants from cell cultures which could then be used to regenerate plants possessing the desired trait. A question important to the utility of this method is whether mutations selected at the biochemical level in cultured cells can be carried to the regenerated plant as a stable trait. Two reports have appeared recently which show that mutations in two tobacco cell lines are stable. Carlson [1 ] selected cells resistant to methionine sulfoximine and grew plr,nts from these cells. Several of these plants exhibited increased free methionine levels and these plants gave some progeny resistant to the methionine analog. Maliga et al. [2] reported the selection of haploid tobacco callus in which growth was resistant to inhibition by streptomycin. Diploid plants were grown from this tissue and the callus formed from leaf sections also showed streptomycin resistance. Likewise, progeny grown from seeds produced by these plants were resistant. 323
Previous repoas from this laboratory [ 3,4] described tobacco and carrot cell mutants resistant to growth inhibition by DL- 5-methyltryptophan, a tryptophan analog. This resistance was due to the presence of an altered anthranilate synthetase which was more resistant to feedback inhibition by the analog, or tryptophan the natural inhibitor. These mutants were obtained from cell lines which had been cultured for many years and had lost the ability to regenerate complete plants. This report describes the selection of 5-methyltryptophan-resistant mutants from freshly initiated carrot cell lines and also describes the passage of this trait from cultured cells to plants and back into cells in culture. Studies concerning the mechanism of resistance are also reported, since the mutants describe~l here were different from those described previously [ 3,4] and did not have a resistant control enzyme. MATERIALS
AND METHODS
Garden carrot root cells (Daucus carota L.) were cultured and the mutant selected as described before [ 3,4] in liquid medium [5]. The DL-forms of all tryptophan analogs were used in the studies. Growth studies were initiated by inoculating 0.5 g fresh weight of suspended cells into 100 ml fresh medium containing the inhibitors at the concentrations listed. After 10 days incubation the cells were collected by vacuum filtration on Mirracloth and weighed. Plants were regenerated by transferring 0.5 ml suspended cells onto 50 ml of the medium described before [5] (lacking 2,4-D, but containing 1 mg/l each of indoleacetic acid and kinetin and 1% agar). Flasks were placed under continuous fluorescent light and after one month small aggregates of greening cells were transferred to fresh medium where complete plants were formed. These plants were placed in soil in pots and grown in the greenhouse. When the plants were about 20 cm tall, 2 cm segments of 1 petiole from each plant were sterilized with 1% sodium hypochlorite for 5 min and placed on regular carrot medium solidified with 1% agar. The callus which formed was used to initiate suspension cultures which were employed for the studies. Anthranilate synthetase activities and free tryptophan were measured as described previously [ 4]. The uptake of filter-sterilized DL- 5-methyltryptophan ( [ 14C ]methyl, 48.2 uCi/umole, CEA, France), DL-5-hydroxytryptophan ([ 3-14C]alanine, 4--7 pCi/umole, Calatomic), DL-tryptophan (2-ringJ4C, 26.7 pCi/pmole, Calatomic) and L-[ULJ4C]leucine (282 ~Ci//~mole, Mallinckrodt) was measured by incubating the cells (which were in the rapid growth phase) in fresh medium containing the labeled compounds at 27 ° . Sterile techniques and solutions were used throughout. At intervals, aliquots of the cell suspensions were collected on filter paper discs by vacuum filtration and rinsed 3 times with 2 ml water containing 0.1 mM of ~ach unlabeled compound. The methods of rinsing and radioabtivity determination were as described by Ferrari and Widholm [6]. At the end of each experiment an aliquot of the incubation medium was plated on nutrient agar plates to detect microbial 324
contamination, but no significant growth was found in the experiments reported here. RESULTS
Cells from a newly initiated garden carrot root culture were ir,oculated into liquid medium containing 220 vM DL-5-methyltryptophan. This concentration completely inhibited growth (increase in fresh weight) when measured after 14 days, but some flasks showed growth within 30 to 60 days. A resistant cell line was established by reinoculating the growing cells into fresh medium containing D L - 5 - m e t h y l t r y p t o p h a n where growth continued. Then, after several subcultures on selective medium, plants were regenerated from both the resistant and normal lines. Cell cultures were re-established from petioles obtained from these regenerated plants. Growth studies with the cultures started from regenerated plants showed that 31 of the 32 cell lines had remained resistant to growth inhibition by 5-methyltryptophan. Of the 4 lines started from plants regenerated from normal cells all were susceptible to growth inhibition. Further studies were carried out with 5 representative cell lines, the origins of which are diagrammed in Fig. 1. Microbial designations are used to describe the mutant lines. All the lines were derived from the original normal line, trp*. This line and the trpP-1 resistant line, which was selected from the trp ÷ line during growth on 5-methyltryptophan, had been in suspension culture continuously for 2 years. Also included was a representative trp ÷ and trpP line obtained from plants {trp ÷2 and trpP-2, respectively) which were grown from the trp ÷ and trpP-1 lines, respectively. Another cell line which had also been selected as resistant to growth inhibition by 5-methyltryptophan was also included. This latter cell line, as will be shown later, differs in the apparent mechanism of resistance and will be denoted trpE-1. The growth curves shown in Figs. 2 and 3 show that the resistant cells, Cell Cultures
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Fig.2. The effect of DL-5-methy|tryptophan on the growth of trp* (~'), trpP-1 (o), trp÷2 (-), lrpP-2 (A) and trpE-1 (~) cultured carrot cells. The study was carried out as described in MATERIALS AND METHODS with the final control weights being 11.7, 12.0, 11.0, 11.6 and 15.3 g, respectively. Fig.3. The effect of Db-5-hydroxytryptophan on the growth of trp* (c), trpP-1 (e), trp÷2 (::), trpP-2 (A) and trpE-I (~) cultured carrot cells. The experimental procedure and the weights of the control cultures were the same as in Fig.2.
whether trpP-1, trpP-2 or trpE-1, are much more resistant to growth inhibition by 5-methyltryptophan or 5-hydroxytryptophan, another tryptophan analog. The 5-hydroxy-analog of tryptophan was somewhat less effective in inhibiting growth of the trp+ cells. The resistant cells were also more resistant to growth inhibition by 5-fluorotryptophan and 6-fluorotryptophan (data not shown) the only other analogs tested. The previously described 5-methyltryptophan-resistant carrot and tobacco cell mutants [ 3,4] possessed anthranilate synthetase activity more resistant to inhibition by 5-methyltryptophan or by tryptophan than was the enzyme activity from the normal cells. Thus, the inhibition of the anthranilate synthetase activities in crude extracts obtained from the carrot cell lines was measured (Figs. 4,5,6) to determine if the same mechanism was operative. It is apparent that the enzyme found in both the trpP-1 and trpP-2 cells was not more resistant to inhibition by tryptophan, 5-methyltryptophan or 5-hydroxytryptophan, unlike the carrot and tobacco mutants described previously [ 3,4]. The cell line designated trpE-1 did have a less sensitive enzyme, but plants have not as yet been grown from this line. None of the 31 trpP-2 cell lines was found to have a feedback insensitive enzyme. The free tryptophan content of these lines was also determined since high levels had been previously found in cells containing the le~s sensitive enzyme [3,4]. In the present study the trp ÷and trp÷2 cells had low levels, 70 326
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.;.-5-HYDROXYTRYPTOPH~N(#M:
Fig.4. Effect o f L-tryptophan on anthranilate synthetase activity in crude extracts from trp ÷ (o), trpP-1 (e), trp ÷2 (~), trpP-2 (A)and trpE-1 (o) cultured carrot cells. Each assay contained 0.2 ml enzyme extract containing 1.38, 1.08, 1.08, 0.88 and 0.76 mg protein and the contrc.ls produced 7.5, 7.5, 11.4, 10.0 and 9.0 nn'loles anthranilate in 30 rain, respectively. Fig.5. Effect of DL-5-methyltryptophan on anthranilate synthetase activity in crude extracts from lrp ÷ (c), trpP-1 (e), trp ÷2 (~), trpP-2 (A)and trpE-1 (u) cultured carrot cells. The extracts and control activities were the same as Fig.4. Fig.6. Effect of DL-5-hydroxytryptophan on anthranilate synthetase activity in crude extracts from trp ÷ (o), trpP-1 (e), trp .2 (:,), trpP-2 (A) and trpE-1 (u) cultured carrot cells. The extracts and control activities were the same as Fig.4.
and 197 nmoles tryptophan/g fresh weight, respectively. The trpP.1 and trpP.2 cells contained 644 and 906 nmoles/g, respectively, while the trpE.1 cells contained 2800. The resistant cells contained significantly higher free tryptophan than the + cells, and the line with the altered enzyme trpE.1 over 3-fold more than the other resistant cells. The 4 other plant trpP-2 lines examined contained from 772 to 962 nmoles/g fresh weight of free tryptophan. Since penetration of the inhibitor into the cells could confer resistance, measurements of the rate of uptake of DL-[ 14 C]-5-methyltryptophan and D L - [ ~ 4 C ] - 5 - h y d r o x y t r y p t o p h a n were made. Both analogs were used since 5-hydroxytryptophan apparently acts in a manner identical to that of 5methyltryptophan [ 5 ]. Fig. 7 shows the uptake of these two analogs and DL-[14C]tryptophan and L-[~4C]leucine by trp ÷ and trpP-1 cells. The uptake of DL- [~4C]-5-methyltryptophan by the trpP-1 cells was less than one-third that by the trp ÷ cells. The uptake of DL-[ 14 C]-5-hydroxytryptophan or DL- [14C]tryptophan by the trpP-1 cells was less than half that of the bp ÷ cells during the 3-h uptake period. The uptake of [~4C] leucine was identical in both cell types. The cells used in these experiments were growing at identical rates in early log phase of growth. Table I lists the results of an experiment comparing the rates of uptake of 327
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2
3
INCUBATION TIME (h) Fig.7. Uptake of ~'C-labeled DL-5- methyltryptophan (SMT), DL-5 -hydroxytryptophan (SHT). D~tryptophan (TRP) and L-leucine (LEU) by trp + (~) and trpP-1 (e) cells. Early exponential growth phase cells (125 mg) were incubated at 27 ° in 25 ml fresh medium containing 1 . C i of each labeled compound. Duplicate I ml samples were removed at time zero and at the times shown from each of 2 duplicate flasks and rinsed with 3 × 2 ml water washes containing 10 -4 M of each unlabeled compound on filter paper discs in a suction flask as described by Ferrari and Widholm [6]. The ~4C in the washed ceils on the filters was determined in a scintillation spectrometer and the standard deviations are denoted by the bars.
TABLE I UPTAKE OF DL- [ '" C ]-5-HYDROXYTRYPTOPHAN BY CULTURED CARROT CELLS Cells (100 mg fresh weight) wert pipetted into 10 ml fresh medium containing 0.1 , C i DL-[ z4C ]-5-hydroxytryptophan and at the times shown, 1-ml aliquots of the cell suspension were removed to determine the uptake as described in MATERIALS AND METHODS. Each point represents one determination. Cell line
t rp + trpP-1 trp÷" trpP-2 trpE-1
328
Uptake of DL- [' 4C ]-5-hydroxytryptophan (cpm/10 mg cells) 3h
6h
97 6 427 1383 456 564
2212 496 2760 931 802
DL- ['4C]-5-hydroxytryptophan by the 5 cell lines being studied (trp ÷, trp+2, trpP-1, trpP-2 and trpE-1). The resistant lines took up much less of the analog at the 3- and 6- h periods than did the + lines. This reduced uptake of 5hydroxytryptophan by the resistant cells was not due to a lower growth rate as no differences in growth rates of the 5 cell lines were noted when grown in a medium without inhibitor. DISCUSSION
The information presented shows that the 5-methyltryptophan-resistance trait can be carried from carrot cell cultures to plants and back into culture. This indicates that this trait is quite stable. No studies on the plants were carried out, except to measure the free tryptophan in the leaves and roots. No reproducible differences in tryptophan levels were noted in plants of trp ÷ or trpP-1 origin. The mechanism of resistance observed with the carrot mutants studied here is, however, apparently different from that observed with both carrot and tobacco mutants described previously [ 3,4 ]. In the tobacco and carrot mutants described previously [ 3,4], the control enzyme, anthranilate synthetase, was less sensitive to feedback inhibition by 5-methyltryptophan or t r y p t o p h a n the natural inhibitor. This resistant enzyme could explain the resistance of these cell lines to growth inhibition by 5-methyltryptophan and also why they accumulate 20 to 30 times the normal free tryptophan. The evidence indicates that the uptake of 5-methyltryptophan by the trpP cell lines, described in this report, is less than one-third that of the trp ~ cells (Fig. 7). The trpP cell lines apparently take up less inhibitor and may in this way be able to grow in the presence of the analogs. Since the cells used were apparently diploid at the time of selection, a permeability mutant may not be expected to be found since both genes coding for the proteins controlling uptake might have to be altered. However, since some of the analog is taken up, the uptake results might be consistent with a heterozygous mutation. Despite the presence of the apparently normal control enzyme, the trpP cells studies here had increased levels of free tryptophan, though not as high as in the cells which had a 5-methyltryptophan resistant anthranilate synthetase (trpE-1). Why the free tryptophan is increased in these trpP cells is not known. This might be explained by a change in membrane permeability which keeps the cellular tryptophan more securely within the cell or more effectively compartmentalized away from the control enzyme. There is evidence indicating that tryptophan is extensively compartmentalized in cultured cells [7 ]. The trpP-1 cells take up less than one-third as much 5-methyltryptophan (Fig. 7), yet require about 30 times as much analog in the medium for equal growth inhibition when compared to trp ÷ cells (Fig. 2). The reason for this difference between uptake and medium inhibitory levels is not known, but more effective compartmentation might play a role.
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Whether t h e D or L forms of 5 - m e t h y l t r y p t o p h a n are t a k e n up and act as ~ inhibitors is likewise n o t known. The L, b u t n o t the V form of t r y p t o p h a n is an effec¢ive inhibitor of anthranilate synthetase from carrot cells, however [ 4]. Tobacco cells can take u p D - t r y p t o p h a n and racemize it enzymatically to L-tryptophan [8 ]. ACKNOWLEDGEMENTS
This research was supported in part b y t h e Illinois Agricultural Experiment Station and was done with t h e technical assistance of Rodger Shewmaker. REFERENCES 1 2 3 4 5 6 7 8
P.S. Carlson, S~ence, 180 (1973) 1366. P. Maliga, A. Sz.-Breznovits and L. Marton, Nature New Biol., 244 (1973) 29. J.M. Widholm, Biochim. Biophys. Acta, 261 (1972) 52. J.M. Widholm, Biochim. Biophys. Acta, 279 (1972)48. J.M. Widholm, Biochim. Biophyso Aeta, 261 (1972) 44. T.E. Ferrari and J.M. Widholm, Anal. Biochem., 56 (1973) 346. J.M. Widholm, Physiol. Plant., 30 (1974) 323. G.A. Miura and S.E. Mills, Plant Physiol., 47 (1971) 483.
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