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Inhibition Of Cell Division By Proline Analogues: Reversal by Proline and High Salinity
Inhibition of Cell Division by Proline Analogues: Reversal by Proline and High Salinity GREGORY C. V ANLERBERGHE and LEWIS M. BROWN Department of Plant Sciences, The University of Western Ontario, London, Ontario N6A 5B7 Canada Received August 20, 1985 . Accepted October 20, 1985
Summary Six proline analogues were tested for their ability to inhibit cell division in N. bacillaris at low salinity. Two analogues, thiazolidine-4-carboxylic acid (T4C) and azetidine-2-carboxylic acid (A2C) were found to be effective inhibitors. Inhibition by 1 mM T4C was not readily reversed by L-proline while inhibition by 20 mM A2C was substantially reversed by an equimolar concentration of L-proline. Also, inhibition by T4C was partially reversed by L-asparagine and Lglutamine and inhibition due to A2C by L-alanine and L-glutamine. When cells were grown in high salinity media, sensitivity to inhibition by T 4C increased. Conversely, A2C became ineffective as an inhibitor of cells at high salinity. Inasmuch as N. bacillaris accummulates high intracellular concentrations of free proline when grown at high salinity, the reversal of inhibition by A2C at high salinity may have been due to the high intracellular pool of proline in such cells.
Key words: Nannochloris bacillaris, analogue, growth inhibition, high salinity, proline.
Introduction The accumulation of organic solutes (osmolytes) in response to osmotic stresses is extremely common in bacteria, plants and animals (Yancey et al., 1982). These osmolytes may act to balance water potential inside and outside the cell. Accumulation of free proline in response to osmotic stress has been well documented in both higher plants (Treichel et al., 1984; Voetberg and Stewart, 1984) and algae (Brown and Hellebust, 1978; Ahmad and Hellebust, 1984). Structural analogues of proline have been shown to have growth inhibitory effects on bacteria (Fowden and Richmond, 1963) higher plants (Kueh and Bright, 1981; Vaughan and Cusens, 1973) and animals (Wasmuth and Caskey, 1976). In many cases, the mechanism of this inhibition may be the incorporation of the analogue rather than proline into protein thereby leading to abnormal proteins (Fowden and Richmond, 1963). Often, proline analogue-mediated growth inhibition can be reversed by addition of proline (Fowden and Richmond, 1963; Kueh and Bright, 1981; Vaughan
Abbreviations ASW, artificial seawater; D-Pro, D-Proline; OH-Pro, hydroxy-L-proline; PIP, L-pipecolic acid; DHP, 3,4-dehydro-L-proline; A2C, L-azetidine-2-carboxylic acid; T4C, L-thiazolidine-4-carboxylic acid; L-Pro, L-proline.
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GREGORY C. VANLERBERGHE and LEWIS M. BROWN
and Cusens, 1973}. Reversal by proline may be due to competition in the cytoplasm between proline and the analogue for incorporation into protein. Also, mutants have been selected which are resistant to growth inhibition by proline analogues due to an increased intracellular proline pool (Cella et al., 1982; Kueh and Bright, 1981, 1982; Kueh et aI., 1984; Nakamori et al., 1982; Wasmuth and Caskey, 1976; Widholm, 1976). In bacteria, there has been recent interest in the relationship between growth inhibition by proline analogues and growth at different salinities since high salinity may result in higher intracellular concentrations of proline (Sugiura and Kisumi, 1985). There has yet, however, to be any report of a reversal of proline analogue-mediated growth inhibition by high salinity. Nannochloris bacilfaris Naumann is a marine, unicellular eucaryotic alga (Chlorophyceae). It is an ideal organism to study the reversability of inhibition by high salinity because it accumulates intracellular free proline to balance extracellular water potential (Brown and Hellebust, 1980; Ancker and Brown, 1985, unpublished). Also, N. bacillaris has no major vacuole (Brown and Elfman, 1983) and therefore the increased proline pool is likely cytoplasmic. The present work reports the inhibition of cell division in N. bacillaris by proline analogues and the ability of proline, other amino acids and high salinity to reverse this inhibition. We believe this to be the first such study in a eucaryotic system.
Materials and Methods The origin and taxonomic status of Nannochloris bacillaris Naumann (isolate UWASH 20-22) has been previously described (Brown, 1982 a; Brown and Elfman, 1983). Culture conditions were as described by Brown (1982 b). The culture was axenic and periodically tested for axenicity (Brown, 1982 a). Cells were grown in an ASW media at various salinities as described (Brown, 1982a). In all cases the cells were pre-adapted at a particular salinity before being used experimentally. Preadaptation was by growth for at least ten transfers (approximately 60 doublings). All media were filter sterilized. Proline analogues used were D-Pro, OH-Pro, PIP, DHP, A2C and T4C. These structures are illustrated in Fig. 1. All amino acids and analogues were from Sigma Chemical Co. Cell concentrations for growth curves and yield determinations were measured with a haemacytometer. In all cases yield was determined after 33 days of growth in the appropriate treatment.
Results
A) Inhibition by proline analogues Four proline analogues, D-Pro, OH-Pro, DHP and PIPdid not substantially affect cell division of N. bacillaris at 10 mM in 7 % ASW while two others, T 4C and A2C produced reduced cell densities (Fig.2). In 7% ASW, 1 mM T4C reduced yield to < 2 % of control while 20 mM A2C reduced yield to 28 % of control by Day 33 (Fig.3). With 20 mM A2C, however, cell density reached a final yield of 71 % of control by about Day 45. When the cells grown for 26 days in media with 20 mM A2C
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were subcultured to fresh media with 20 mM A2e there was no further growth inhibition, although there was a short lag phase (due to the age of the culture) (Fig. 4).
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GREGORY
C. VANLERBERGHE and
LEWIS
M. BROWN
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TIME (DAYS) Fig. 3: Log cells/ml vs. time for N badllaris in 7% ASW (circles), 7% ASW with ImM T4C (squares) and 7% ASW with 20 mM A2C (triangles). All values are average of 3 determinations ± SD. In many cases the symbol size is larger than the standard deviation bars.
B) Ability ofL-Proline to reverse the inhibition by T4C and A2C The addition of 20 mM or 100 mM L-Pro to the medium was unable to reverse the inhibition caused by 1 mM T4C in 7% ASW (Table 1). Also, a range of concentrations of i-Pro between 1 mM and 100 mM was unable to reverse inhibition by T4C in 7%,100% or 200% ASW (data not shown). Yield in each case was always less than 2 % of the control. Conversely, inhibition in 20 mM A2C could be substantially reversed by an equimolar concentration of i-Pro (Table 1). AS: 1 concentration of i-Pro to A2C completely reversed the inhibition (Table 1).
C) Ability ofgrowth at high salinity to reverse T4C and A2C inhibition T4C was found to be an effective inhibitor of cell division for cells growing in 7%, 100 % and 200 % ASW. Growth at high salinity made the cells more susceptible to inhibition by T4C {Fig. 5). The T4C concentration required for a 50% reduction in yield decreased from .77 mM in 7 % ASW to .52 mM in 100 % ASW and to .22 mM in 200 % ASW (Fig. 5).
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Fig. 4: Log cells/ ml vs. time for N. bacilfaris in 7 % ASW (circles, triangles) and 7 % ASW with 20 mM A2C (squares). In two experiments (triangles, squares, dashed line) cells were preadapted for 26 days to 20 mM A2e. Cell division rates and yield were similar regardless of whether these cells were grown in the presence (squares) or absence (triangles) of A2e. These pre-adapted cells are represented by the dashed line (they showed a short lag phase reflecting their origin from a stationary phase culture: 26 days old). Those without A2C exposure (circles, solid line) exhibited no lag. All values are means of 3 determinations ± SD. In many cases the symbol size is larger than the standard deviation bars. Table 1: Ability of L-Pro to reverse the growth inhibition by T4C and A2C of N. bacilfaris in 7% ASW. Control had no inhibitor added. Values are the mean of 2 determinations. Control yield was 8.0 ± 0.4 x 107 cells· ml- 1 for the T 4C treatments and 8.7 ±0.5 x 107 cells· ml- 1 for the A2C treatments (N = 4). Treatment lmM T4C 1 mM T4C+ 20mM L-Pro 1 mM T 4C + 100 mM L-Pro 20mMA2C 20 mM A2C + 20 mM L-Pro 20 mM A2C + 100 mM L-Pro
Yield (% of control) 1.0
2.9 0.9*) 29.0 56.5 107.0*)
*) single determinations.
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GREGORY
e. VANLERBERGHE and
LEWIS
M.
BROWN
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Fig. 5: Yield (% of control) of N. bacilla· ris in 7% (circles), 100% (triangles) and 200 % (squares) ASW supplemented by different concentrations of T 4C. Each salinity had its own control (no HC) but all controls were set to a yield of 100 %. Control yields were 8.5±0.5x107 cells'ml- I (7% ASW), 9.9±0.5x107 cells·ml- I (100% ASW) and 7.8±0.8x107 cells·ml- I (200% ASW) (N = 4).
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Fig. 6: Yield (% of control) of N. bacillaris in 7 % (circles) 100 % (triangles) and 200 % (squares) ASW supplemented by different concentrations of A2e. Each salinity had its own control (no A2C) but all controls were set to a yield of 100 %. Control yields were 8.0 ±0.6 x 107 cells' ml- I (7% ASW), 1.1±0.6x 108 cells' ml- I (100% ASW) and 7.0±0.5x 107 cells' ml- I (200% ASW).
There was no inhibition of cell division by A2C at high salinity. While 20 roM A2C reduced yield of cells of 7 % ASW to 21 % of control, there was no such reduction for
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cells growing at 100 % and 200 % ASW {Fig. 6). In fact, when cells were grown in 100 % and 200 % ASW they withstood very high concentrations of A2C without any apparent inhibition of growth {Fig. 6). Particularly at 200 % ASW it appears that A2C actually increased yield by Day 33.
D) Ability of other amino acids to reverse T4G and A2Gmediated inhibition Other amino acids were tested for their ability to reverse the inhibition by 1 mM T 4C or 20 mM A2e. Asparagine and glutamine were able to substantially reverse the inhibition by 1 mM T4C (Table 2). Glutamine and alanine were able to partially reverse the inhibition by 20 mM A2e (Table 2). Most other amino acids had little effect or increased the inhibition. Table 2: Yield (% of control) of N. bacillaris in 7% ASW supplemented with T4C or A2C plus various amino acids. Control had no amino acid or analogue added. Control yield was 9.2±0.5xl07 cells·ml- 1 for the T4C treatments and 8.1±0.9xl07 cells·ml- 1 for the A2C treatments (N = 4). Treatment*) no addition L-alanine L-arginine L-asparagine L-glutamic acid L-glutamine glycine L-isoleucine L-Ieucine L-Iysine L-methionine OH-Pro L-ornithine L-phenylalanine D-Pro L-serine L-threonine L-tyrosine L-valine
Yield (% of control) T4C
A2C
0.2 0.4 1.0 61.8 0.0 32.0 0.8
28.6 71.4 10.8 15.6 12.1 70.1 27.3 N.D.**) N.D. N.D. N.D. 31.2 N.D. N.D. N.D. N.D. 15.6 N.D. 29.9
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0.0 0.0 0.02 6.4 0.0 0.03 1.6 0.5 9.6 0.6 2.6
*) All treatments contained 1 mM T 4C or 20 mM A2e. All amino acids were at a concentration of 20 mM except L-glutamic acid and L-tyrosine which were 1 mM. "") Not determined. In experiments to determine the effect of the various amino acids on cell division it was found that most did not interfere with cell division (data not shown) except 20 mM L-glutamic acid, 20 mM L-tryptophan, 20 mM L-histidine and 20 mM L-aspartie acid which were toxic.
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C. VANLERBERGHE and LEWIS M. BROWN
Discussion Of the six proline analogues tested T 4C and AlC were found to be the most effective inhibitors of cell division in N bacillaris in 7 % ASW (Fig. 2). T 4C was the most effective growth inhibitor in that much lower concentrations of the analogue were required (Table 1, Figs. 5, 6). The characteristics of inhibition by T4C and A2C were different in that, after about 15 days, N bacillaris seemed to overcome AlC inhibition somewhat, whereas this was not seen with T4C inhibition (Fig. 3). The fact that cell division in N bacillaris, after long-term exposure to 20 mM A2C was no longer inhibited by 20 mM AlC (Fig. 3, 4) suggests that there has either been some metabolic adaptation or selection for some members of the population more resistant to A2e. Since T4C was an effective inhibitor in 7 %, 100 % and 200 % ASW media (Fig. 5), it appears that the high intracellular pool of proline in these cells when grown at high salinity (Brown and Hellebust, 1980) does not reduce their sensitivity to T4e. In fact the sensitivity to T4C increased with increasing salinity. T4C has been shown to be an effective inhibitor of proline oxidation in isolated barley mitochondria, and in wilted leaves which rapidly synthesize proline, T4C inhibited proline synthesis (Elthon and Stewart, 1984). Also, T4C inhibited pyrroline5-carboxylic acid reductase (the last enzyme in the synthesis of proline) from soybean leaves (Miler and Stewart, 1976) while no such inhibition was seen in barley (Elthon and Stewart, 1984). If T4C is able to disrupt proline metabolism in N bacillans then it is not surprising that sensitivity to T 4C may increase during high salinity since this alga accumulates intracellular free proline to partially balance extracellular water potential during osmotic stress (Brown and Hellebust, 1980). A continuing need for high rates of proline biosynthesis in these exponential phase cultures is therefore suggested. T 4C may interfere with these high rates of biosynthesis. One cannot exclude the possibility, however, that there is some other type of synergistic effect of T 4C and high salinity on growth in that both treatments are presumably placing stress on the cell. Inhibition due to T 4C was not reversed by the high intracellular proline pool in cells grown at high salinity, nor could it be readily reversed by proline added to the media (Table 1). Similarly, Widholm (1976) found that proline could not reverse T4C inhibition of carrot cell growth while growth inhibition by OH-Pro and AlC could be reversed by proline. In another study (Kueh and Bright, 1981), inhibition of the growth of mature barley embryos by T 4C was reversed by proline however the degree of reversal is not known. It may be that T 4C is not an effective functional analogue of proline in some biological systems. In barley (Elthon and Stewart, 1984) it was found that T4C did not affect the incorporation of proline into protein suggesting that it cannot replace proline during protein biosynthesis. However, T4C did act as a substrate for a purified prolyl-transfer RNA synthetases from Phaseolus aureus, although it was activated at only 2 % the rate of proline (Peterson and Fowden, 1965). However, T4C protected Pro-tRNA synthetase against thermal denaturation and in-
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hibited incorporation of 14C- or 3H-Proline into tRNA (Norris and Fowden, 1972). It would appear, therefore, that T4C is a functional proline analogue only in certain enzyme systems. The inhibition by A2C could be reversed by both high salinity and proline (Table 1, Fig. 6). In other organisms, mutants have been obtained which are resistant to growth inhibition by A2C by being overproducers of proline (Riccardi et al., 1981; Cella et al., 1982; Lodato et al., 1984). It is suggested therefore that the reversal of A2C inhibition by high salinity in N. bacillaris may be due to the high intracellular proline pool reported for such cells (Brown and Hellebust, 1980). Previously, it was found that A2C severely inhibited the induction of nitrate reductase by nitrate in plants grown under conditions of nitrate starvation (Hewitt and Notton, 1967). However, induction of the enzyme in response to molybdenum was less inhibited in tissues of molybdenum deficient plants which had a high endogenous proline content. Alternatively, there may be some other explanation than the high intracellular proline pool for the insensitivity to A2C at high salinity. Possibly, there may be a salinity dependent change in the transport system responsible for A2C uptake. A carrot cell line was found to have decreased A2C uptake due to an artificially expanded amino acid pool (Cella et al., 1982). They hypothesized that the carrier{s) for amino acids may have been saturated due to the expansion of the amino acid pool and therefore less available for the uptake of amino acids and! or A2e. Previous work (Brown and Hellebust, 1980) has shown that there is an increase at high salinity of four amino acids (alanine, aspartic acid, glutamic acid, proline) in N. bacillaris. There may be decreased A2C uptake by N. bacillaris at high salinity due to a high intracellular amino acid pool preventing A2C uptake. There was no general reversal of amino acid toxicity at high salinity because 20 mM L-glutamic acid and 20 mM L-aspartic acid (which we found to be toxic to N. bacillaris in 7 % ASW) was also toxic to N. bacillaris in 200% ASW (data not shown). Replacement of proline by A2C in newly synthesized protein has been shown in bacteria (Fowden and Richmond, 1963) algae (Troxler and Brown, 1974) and higher plants (Fowden and Richmond, 1963; Cella et al., 1982). The presence of such a residue results in altered protein structure and thus leads to growth inhibitory effects on the organism. Also, reversal of A2C growth inhibition by the addition of proline has been shown (Vaughan and Cusens, 1973; Widholm, 1976; Kueh and Bright, 1981). One consequence of A2C incorporation into protein was an inhibition of chlorophyll a and phycocyanin synthesis in cells of the unicellular alga Cyanidium caldarium (Troxler and Brown, 1974). They also found that proline was able to partially reverse the inhibition. The mechanism by which T 4C and A2C growth inhibition could be reversed by other amino acids (Table2) is not known. In higher plants (Reinhold and Kaplan, 1984) and algae (Reinhold and Kaplan, 1984; Sauer, 1984) amino acid transport systems have been reported which have affinities for all or groups of amino acids. In
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Chlorella vulgaris, growth inhibition by the methionine analog methionine sulfoximine could be reversed by glutamine because both glutamine and methionine sulfoximine competed for a common uptake system (Meins and Abrams, 1972). It is POSsible therefore that the mechanism by which some amino acids can reverse T 4C and A2C inhibition is by competition for uptake by a common transport system. In prokaryotes it has been reported that sensitivity to proline analogues increased in media of elevated osmolarity (Csonka, 1982; Sugiura and Kisumi, 1985). For example, in Salmonella typhimurium this was attributed to an additional proline permease which functions only in media of elevated osmolarity (Csonka, 1982) and served to accumulate higher levels of the inhibitors. At low salinity a proline producing strain of Serratia marcescans was completely resistant to 10mM A2C, T4C and DHP. However, in the presence of 500 mM NaCl, 1 mM A2C or DHP significantly inhibited growth (Sugiura and Kisumi, 1985). In contrast to previous reports with procaryotic organisms, the present work represents the first example of a reversal of proline analogue-mediated inhibition by high salinity. This reversal of inhibition by A2C is most likely to be due to the high intracellular proline pool in these cells. If the mechanism of inhibition by A2C in this alga is by incorporation into protein as has been found in other organisms (Fowden and Richmond, 1963; Troxler and Brown, 1974; Cella et aI., 1982), then the reversal of A2C growth inhibition by the high intracellular proline pool at high salinity suggests that this cytoplasmic pool may be available for protein synthesis. Acknowledgements Financial support was provided by the Natural Sciences and Engineering Research Council of Canada. References AHMAD, 1. and J. A. HELLEBUST: Osmoregulation in the extremely euryhaline marine micro-alga Chlorella autotrophica. Plant Physiol. 74, 1010-1015 (1984). BROWN, L. M.: Production of axenic cultures of algae by an osmotic method. Phycologia 21, 408-410 (1982 a). - Photosynthetic and growth responses to salinity in a marine isolate of Nannochloris baei/· laris (Chlorophyceae). J. Phycol. 18, 483 - 488 (1982 b). BROWN, L. M. and B. ELFMAN: Is autosporulation a feature of Nannochloris? Can. J. Bot. 61, 2647 -2657 (1983). BROWN, L. M. and J. A. HaLEBUST: Sorbitol and proline as intracellular osmotic solutes in the green alga Stichococcus bacillaris. Can. J. Bot. 56, 676-679 (1978). - - The contribution of organic solutes to osmotic balance in some green and eustigmatophyte algae. J. Phycol. 16, 265-270 (1980). CELLA, R., B. PARISI, and E. NIELSEN: Characterization of a carrot cell line resistant to azetidine2-carboxylic acid. Plant Sci. Lett. 24, 125-135 (1982). CSONKA, L. N.: A third L-proline permease in Salmonella typhimurium which functions in media of elevated osmotic strength. J. Bacteriol. 151, 1433-1443 (1982). ELTHON, T. E. and C. R. STEWART: Effects of the proline analog L-thiazolidine-4-carboxylic acid on proline metabolism. Plant Physiol. 74, 213-218 (1984).
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FOWDEN, L. and M. H. RICHMOND: Replacement of proline by azetidine-2-carboxylic acid during biosynthesis of protein. Biochim. Biophys. Acta 71, 459-461 (1963). HEWITT, E. J. and B. A. NOTTON: Inhibition by L-azetidine-2-carboxylic acid of induction of nitrate reductase in plants and its reversal by L-proline. Phytochemistry 6, 1329-1335 (1967). KUEH, J. S. H. and W. J. BRIGHT: Proline accumulation in a barley mutant resistant of trans-4hydroxyl-L-proline. Planta 153,166-171 (1981). - - Biochemical and genetical analysis of 3 proline-accumulating barley mutants. Plant Sci. Lett. 27, 233-241 (1982). KUEH, J. S. H., J. M. HILL, S. J. SMITH, and S. W. J. BRIGHT: Proline biosynthesis in a proline-accumulating barley mutant. Phytochemistry 23, 2207 -2210 (1984). LODATO, R. F., R. J. SMITH, D. L. VALLE, and K. CRANE: Mutant cell lines resistant to azetidine-2carboxylic acid: Alterations in the synthesis of proline from glutamic acid. J. Cell. Physiol. 119, 137 -143 (1984). MEINS, F. and M. A. ABRAMS: How methionine and glutamine prevent inhibition of growth by methionine sulfoximine. Biochim. Biophys. Acta 266, 307 - 311 (1972). MILER, P. M. and C. R. STEWART: Pyrroline-5-carboxylic acid reductase from soybean leaves. Phytochemistry 15, 1855-1857 (1976). NAKAMORI, S., H. MORIOKA, F. YOSHIMAGA, and S. YAMANAKA: Fermentative production of Lproline of DL-3,4-dehydroproline resistant mutants of L-glutamate producing bacteria. Agric. BioI. Chern. 46, 487 -491 (1982). NORRIS, R. D. and L. FOWDEN: Substrate discrimination by prolyl-tRNA synthetase from various higher plants. Phytochemistry 11, 2921-2935 (1972). PETERSON, P. J. and L. FOWDEN: Purification properties and comparative specificities of the enzyme prolyl-transfer ribonucleic acid synthetase from Phaseolus aureus and Polygonatum multiflorum. Biochem. J. 97, 112-124 (1965). REINHOLD, L. and A. KAPLAN: Membrane transport of sugars and amino acids. Ann. Rev. Plant. Physiol. 35,45-83 (1984). RiCCARDI, G., S. SORA, and O. CIFFERRI: Production of amino acids by analog-resistant mutants of the Cyanobacterium Spirulina platensis. J. Bacteriol. 147, 1002-1007 (1981). SAUER, N.: A general amino-acid permease is inducible in Chlorella vulgaris. Planta 161, 425431 (1984). SUGIURA, M. and M. KISUMI: Proline-hyperproducing strains of Seratia marcescens: Enhancement of proline analog-mediated growth inhibition by increasing osmotic stress. App!. Environ. Microbiol. 49, 782-786 (1985). TREICHEL, S., E. BRINCKMANN, B. SCHEITLER, and D. J. VON WILLERT: Occurrence and changes of proline content in plants in the southern Namib Desert in relation to increasing and decreasing drought. Planta 162, 236-242 (1984). TROXLER, R. F. and A. S. BROWN: Metabolism of L-azetidine-2-carboxylic acid in the alga Cyani· dium caldarium. Biochim. Biophys. Acta 366, 341-349 (1974). VAUGHAN, D. and E. CUSENS: Effects of hydroxyproline on the growth of excised root segments of Pisum sativum under aseptic conditions. Planta 112, 243 -252 (1973). VOETBERG, G. and C. R. STEWART: Steady state proline levels in salt-shocked barley leaves. Plant Physiol. 76, 567 - 570 (1984). WASMUTH, J. J. and C. T. CASKEY: Biochemical characterization of azetidine carboxylic acid-resistant Chinese hamster cells. Cell 8, 71-77 (1976). WIDHOLM, J. M.: Selection and characterization of cultured carrot and tobacco cells resistant to lysine, methionine and proline analogs. Can. J. Bot. 54, 1523-1529 (1976). YANCEY, P. H., M. E. CLARK, S. C. HAND, R. D. BOWLUS, and G. V. SOMERO: Living with water stress. Evolution of osmolyte systems. Science 217, 1214-1222 (1982).
]. Plant Physiol. Vol. 123. pp. 229- 239 (1986)
Summary Six proline analogues were tested for their ability to inhibit cell division in N. bacillaris at low salinity. Two analogues, thiazolidine-4-carboxylic acid (T4C) and azetidine-2-carboxylic acid (A2C) were found to be effective inhibitors. Inhibition by 1 mM T4C was not readily reversed by L-proline while inhibition by 20 mM A2C was substantially reversed by an equimolar concentration of L-proline. Also, inhibition by T4C was partially reversed by L-asparagine and Lglutamine and inhibition due to A2C by L-alanine and L-glutamine. When cells were grown in high salinity media, sensitivity to inhibition by T 4C increased. Conversely, A2C became ineffective as an inhibitor of cells at high salinity. Inasmuch as N. bacillaris accummulates high intracellular concentrations of free proline when grown at high salinity, the reversal of inhibition by A2C at high salinity may have been due to the high intracellular pool of proline in such cells.
Key words: Nannochloris bacillaris, analogue, growth inhibition, high salinity, proline.
Introduction The accumulation of organic solutes (osmolytes) in response to osmotic stresses is extremely common in bacteria, plants and animals (Yancey et al., 1982). These osmolytes may act to balance water potential inside and outside the cell. Accumulation of free proline in response to osmotic stress has been well documented in both higher plants (Treichel et al., 1984; Voetberg and Stewart, 1984) and algae (Brown and Hellebust, 1978; Ahmad and Hellebust, 1984). Structural analogues of proline have been shown to have growth inhibitory effects on bacteria (Fowden and Richmond, 1963) higher plants (Kueh and Bright, 1981; Vaughan and Cusens, 1973) and animals (Wasmuth and Caskey, 1976). In many cases, the mechanism of this inhibition may be the incorporation of the analogue rather than proline into protein thereby leading to abnormal proteins (Fowden and Richmond, 1963). Often, proline analogue-mediated growth inhibition can be reversed by addition of proline (Fowden and Richmond, 1963; Kueh and Bright, 1981; Vaughan
Abbreviations ASW, artificial seawater; D-Pro, D-Proline; OH-Pro, hydroxy-L-proline; PIP, L-pipecolic acid; DHP, 3,4-dehydro-L-proline; A2C, L-azetidine-2-carboxylic acid; T4C, L-thiazolidine-4-carboxylic acid; L-Pro, L-proline.
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GREGORY C. VANLERBERGHE and LEWIS M. BROWN
and Cusens, 1973}. Reversal by proline may be due to competition in the cytoplasm between proline and the analogue for incorporation into protein. Also, mutants have been selected which are resistant to growth inhibition by proline analogues due to an increased intracellular proline pool (Cella et al., 1982; Kueh and Bright, 1981, 1982; Kueh et aI., 1984; Nakamori et al., 1982; Wasmuth and Caskey, 1976; Widholm, 1976). In bacteria, there has been recent interest in the relationship between growth inhibition by proline analogues and growth at different salinities since high salinity may result in higher intracellular concentrations of proline (Sugiura and Kisumi, 1985). There has yet, however, to be any report of a reversal of proline analogue-mediated growth inhibition by high salinity. Nannochloris bacilfaris Naumann is a marine, unicellular eucaryotic alga (Chlorophyceae). It is an ideal organism to study the reversability of inhibition by high salinity because it accumulates intracellular free proline to balance extracellular water potential (Brown and Hellebust, 1980; Ancker and Brown, 1985, unpublished). Also, N. bacillaris has no major vacuole (Brown and Elfman, 1983) and therefore the increased proline pool is likely cytoplasmic. The present work reports the inhibition of cell division in N. bacillaris by proline analogues and the ability of proline, other amino acids and high salinity to reverse this inhibition. We believe this to be the first such study in a eucaryotic system.
Materials and Methods The origin and taxonomic status of Nannochloris bacillaris Naumann (isolate UWASH 20-22) has been previously described (Brown, 1982 a; Brown and Elfman, 1983). Culture conditions were as described by Brown (1982 b). The culture was axenic and periodically tested for axenicity (Brown, 1982 a). Cells were grown in an ASW media at various salinities as described (Brown, 1982a). In all cases the cells were pre-adapted at a particular salinity before being used experimentally. Preadaptation was by growth for at least ten transfers (approximately 60 doublings). All media were filter sterilized. Proline analogues used were D-Pro, OH-Pro, PIP, DHP, A2C and T4C. These structures are illustrated in Fig. 1. All amino acids and analogues were from Sigma Chemical Co. Cell concentrations for growth curves and yield determinations were measured with a haemacytometer. In all cases yield was determined after 33 days of growth in the appropriate treatment.
Results
A) Inhibition by proline analogues Four proline analogues, D-Pro, OH-Pro, DHP and PIPdid not substantially affect cell division of N. bacillaris at 10 mM in 7 % ASW while two others, T 4C and A2C produced reduced cell densities (Fig.2). In 7% ASW, 1 mM T4C reduced yield to < 2 % of control while 20 mM A2C reduced yield to 28 % of control by Day 33 (Fig.3). With 20 mM A2C, however, cell density reached a final yield of 71 % of control by about Day 45. When the cells grown for 26 days in media with 20 mM A2C
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(""N~O
U
Fig. 1: Structure of L-Proline and its analogues.
L-PRO
D-PRO
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60
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Fig. 2: Yield (% of control) of N. baeil· laris in 7 % ASW supplemented with different proline analogues. Each analogue was at a concentration of 10 mM. Control yield was 9.2±0.8 x 107 cells·ml- 1 (N = 4).
*t:;
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I
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were subcultured to fresh media with 20 mM A2e there was no further growth inhibition, although there was a short lag phase (due to the age of the culture) (Fig. 4).
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GREGORY
C. VANLERBERGHE and
LEWIS
M. BROWN
8.0
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5
10
15
20
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TIME (DAYS) Fig. 3: Log cells/ml vs. time for N badllaris in 7% ASW (circles), 7% ASW with ImM T4C (squares) and 7% ASW with 20 mM A2C (triangles). All values are average of 3 determinations ± SD. In many cases the symbol size is larger than the standard deviation bars.
B) Ability ofL-Proline to reverse the inhibition by T4C and A2C The addition of 20 mM or 100 mM L-Pro to the medium was unable to reverse the inhibition caused by 1 mM T4C in 7% ASW (Table 1). Also, a range of concentrations of i-Pro between 1 mM and 100 mM was unable to reverse inhibition by T4C in 7%,100% or 200% ASW (data not shown). Yield in each case was always less than 2 % of the control. Conversely, inhibition in 20 mM A2C could be substantially reversed by an equimolar concentration of i-Pro (Table 1). AS: 1 concentration of i-Pro to A2C completely reversed the inhibition (Table 1).
C) Ability ofgrowth at high salinity to reverse T4C and A2C inhibition T4C was found to be an effective inhibitor of cell division for cells growing in 7%, 100 % and 200 % ASW. Growth at high salinity made the cells more susceptible to inhibition by T4C {Fig. 5). The T4C concentration required for a 50% reduction in yield decreased from .77 mM in 7 % ASW to .52 mM in 100 % ASW and to .22 mM in 200 % ASW (Fig. 5).
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•
8.0
-----II '....I
::2:
en ....I
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TIME (DAYS)
Fig. 4: Log cells/ ml vs. time for N. bacilfaris in 7 % ASW (circles, triangles) and 7 % ASW with 20 mM A2C (squares). In two experiments (triangles, squares, dashed line) cells were preadapted for 26 days to 20 mM A2e. Cell division rates and yield were similar regardless of whether these cells were grown in the presence (squares) or absence (triangles) of A2e. These pre-adapted cells are represented by the dashed line (they showed a short lag phase reflecting their origin from a stationary phase culture: 26 days old). Those without A2C exposure (circles, solid line) exhibited no lag. All values are means of 3 determinations ± SD. In many cases the symbol size is larger than the standard deviation bars. Table 1: Ability of L-Pro to reverse the growth inhibition by T4C and A2C of N. bacilfaris in 7% ASW. Control had no inhibitor added. Values are the mean of 2 determinations. Control yield was 8.0 ± 0.4 x 107 cells· ml- 1 for the T 4C treatments and 8.7 ±0.5 x 107 cells· ml- 1 for the A2C treatments (N = 4). Treatment lmM T4C 1 mM T4C+ 20mM L-Pro 1 mM T 4C + 100 mM L-Pro 20mMA2C 20 mM A2C + 20 mM L-Pro 20 mM A2C + 100 mM L-Pro
Yield (% of control) 1.0
2.9 0.9*) 29.0 56.5 107.0*)
*) single determinations.
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GREGORY
e. VANLERBERGHE and
LEWIS
M.
BROWN
100
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Fig. 5: Yield (% of control) of N. bacilla· ris in 7% (circles), 100% (triangles) and 200 % (squares) ASW supplemented by different concentrations of T 4C. Each salinity had its own control (no HC) but all controls were set to a yield of 100 %. Control yields were 8.5±0.5x107 cells'ml- I (7% ASW), 9.9±0.5x107 cells·ml- I (100% ASW) and 7.8±0.8x107 cells·ml- I (200% ASW) (N = 4).
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100
120
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160
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Fig. 6: Yield (% of control) of N. bacillaris in 7 % (circles) 100 % (triangles) and 200 % (squares) ASW supplemented by different concentrations of A2e. Each salinity had its own control (no A2C) but all controls were set to a yield of 100 %. Control yields were 8.0 ±0.6 x 107 cells' ml- I (7% ASW), 1.1±0.6x 108 cells' ml- I (100% ASW) and 7.0±0.5x 107 cells' ml- I (200% ASW).
There was no inhibition of cell division by A2C at high salinity. While 20 roM A2C reduced yield of cells of 7 % ASW to 21 % of control, there was no such reduction for
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cells growing at 100 % and 200 % ASW {Fig. 6). In fact, when cells were grown in 100 % and 200 % ASW they withstood very high concentrations of A2C without any apparent inhibition of growth {Fig. 6). Particularly at 200 % ASW it appears that A2C actually increased yield by Day 33.
D) Ability of other amino acids to reverse T4G and A2Gmediated inhibition Other amino acids were tested for their ability to reverse the inhibition by 1 mM T 4C or 20 mM A2e. Asparagine and glutamine were able to substantially reverse the inhibition by 1 mM T4C (Table 2). Glutamine and alanine were able to partially reverse the inhibition by 20 mM A2e (Table 2). Most other amino acids had little effect or increased the inhibition. Table 2: Yield (% of control) of N. bacillaris in 7% ASW supplemented with T4C or A2C plus various amino acids. Control had no amino acid or analogue added. Control yield was 9.2±0.5xl07 cells·ml- 1 for the T4C treatments and 8.1±0.9xl07 cells·ml- 1 for the A2C treatments (N = 4). Treatment*) no addition L-alanine L-arginine L-asparagine L-glutamic acid L-glutamine glycine L-isoleucine L-Ieucine L-Iysine L-methionine OH-Pro L-ornithine L-phenylalanine D-Pro L-serine L-threonine L-tyrosine L-valine
Yield (% of control) T4C
A2C
0.2 0.4 1.0 61.8 0.0 32.0 0.8
28.6 71.4 10.8 15.6 12.1 70.1 27.3 N.D.**) N.D. N.D. N.D. 31.2 N.D. N.D. N.D. N.D. 15.6 N.D. 29.9
1.3
0.0 0.0 0.02 6.4 0.0 0.03 1.6 0.5 9.6 0.6 2.6
*) All treatments contained 1 mM T 4C or 20 mM A2e. All amino acids were at a concentration of 20 mM except L-glutamic acid and L-tyrosine which were 1 mM. "") Not determined. In experiments to determine the effect of the various amino acids on cell division it was found that most did not interfere with cell division (data not shown) except 20 mM L-glutamic acid, 20 mM L-tryptophan, 20 mM L-histidine and 20 mM L-aspartie acid which were toxic.
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Discussion Of the six proline analogues tested T 4C and AlC were found to be the most effective inhibitors of cell division in N bacillaris in 7 % ASW (Fig. 2). T 4C was the most effective growth inhibitor in that much lower concentrations of the analogue were required (Table 1, Figs. 5, 6). The characteristics of inhibition by T4C and A2C were different in that, after about 15 days, N bacillaris seemed to overcome AlC inhibition somewhat, whereas this was not seen with T4C inhibition (Fig. 3). The fact that cell division in N bacillaris, after long-term exposure to 20 mM A2C was no longer inhibited by 20 mM AlC (Fig. 3, 4) suggests that there has either been some metabolic adaptation or selection for some members of the population more resistant to A2e. Since T4C was an effective inhibitor in 7 %, 100 % and 200 % ASW media (Fig. 5), it appears that the high intracellular pool of proline in these cells when grown at high salinity (Brown and Hellebust, 1980) does not reduce their sensitivity to T4e. In fact the sensitivity to T4C increased with increasing salinity. T4C has been shown to be an effective inhibitor of proline oxidation in isolated barley mitochondria, and in wilted leaves which rapidly synthesize proline, T4C inhibited proline synthesis (Elthon and Stewart, 1984). Also, T4C inhibited pyrroline5-carboxylic acid reductase (the last enzyme in the synthesis of proline) from soybean leaves (Miler and Stewart, 1976) while no such inhibition was seen in barley (Elthon and Stewart, 1984). If T4C is able to disrupt proline metabolism in N bacillans then it is not surprising that sensitivity to T 4C may increase during high salinity since this alga accumulates intracellular free proline to partially balance extracellular water potential during osmotic stress (Brown and Hellebust, 1980). A continuing need for high rates of proline biosynthesis in these exponential phase cultures is therefore suggested. T 4C may interfere with these high rates of biosynthesis. One cannot exclude the possibility, however, that there is some other type of synergistic effect of T 4C and high salinity on growth in that both treatments are presumably placing stress on the cell. Inhibition due to T 4C was not reversed by the high intracellular proline pool in cells grown at high salinity, nor could it be readily reversed by proline added to the media (Table 1). Similarly, Widholm (1976) found that proline could not reverse T4C inhibition of carrot cell growth while growth inhibition by OH-Pro and AlC could be reversed by proline. In another study (Kueh and Bright, 1981), inhibition of the growth of mature barley embryos by T 4C was reversed by proline however the degree of reversal is not known. It may be that T 4C is not an effective functional analogue of proline in some biological systems. In barley (Elthon and Stewart, 1984) it was found that T4C did not affect the incorporation of proline into protein suggesting that it cannot replace proline during protein biosynthesis. However, T4C did act as a substrate for a purified prolyl-transfer RNA synthetases from Phaseolus aureus, although it was activated at only 2 % the rate of proline (Peterson and Fowden, 1965). However, T4C protected Pro-tRNA synthetase against thermal denaturation and in-
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hibited incorporation of 14C- or 3H-Proline into tRNA (Norris and Fowden, 1972). It would appear, therefore, that T4C is a functional proline analogue only in certain enzyme systems. The inhibition by A2C could be reversed by both high salinity and proline (Table 1, Fig. 6). In other organisms, mutants have been obtained which are resistant to growth inhibition by A2C by being overproducers of proline (Riccardi et al., 1981; Cella et al., 1982; Lodato et al., 1984). It is suggested therefore that the reversal of A2C inhibition by high salinity in N. bacillaris may be due to the high intracellular proline pool reported for such cells (Brown and Hellebust, 1980). Previously, it was found that A2C severely inhibited the induction of nitrate reductase by nitrate in plants grown under conditions of nitrate starvation (Hewitt and Notton, 1967). However, induction of the enzyme in response to molybdenum was less inhibited in tissues of molybdenum deficient plants which had a high endogenous proline content. Alternatively, there may be some other explanation than the high intracellular proline pool for the insensitivity to A2C at high salinity. Possibly, there may be a salinity dependent change in the transport system responsible for A2C uptake. A carrot cell line was found to have decreased A2C uptake due to an artificially expanded amino acid pool (Cella et al., 1982). They hypothesized that the carrier{s) for amino acids may have been saturated due to the expansion of the amino acid pool and therefore less available for the uptake of amino acids and! or A2e. Previous work (Brown and Hellebust, 1980) has shown that there is an increase at high salinity of four amino acids (alanine, aspartic acid, glutamic acid, proline) in N. bacillaris. There may be decreased A2C uptake by N. bacillaris at high salinity due to a high intracellular amino acid pool preventing A2C uptake. There was no general reversal of amino acid toxicity at high salinity because 20 mM L-glutamic acid and 20 mM L-aspartic acid (which we found to be toxic to N. bacillaris in 7 % ASW) was also toxic to N. bacillaris in 200% ASW (data not shown). Replacement of proline by A2C in newly synthesized protein has been shown in bacteria (Fowden and Richmond, 1963) algae (Troxler and Brown, 1974) and higher plants (Fowden and Richmond, 1963; Cella et al., 1982). The presence of such a residue results in altered protein structure and thus leads to growth inhibitory effects on the organism. Also, reversal of A2C growth inhibition by the addition of proline has been shown (Vaughan and Cusens, 1973; Widholm, 1976; Kueh and Bright, 1981). One consequence of A2C incorporation into protein was an inhibition of chlorophyll a and phycocyanin synthesis in cells of the unicellular alga Cyanidium caldarium (Troxler and Brown, 1974). They also found that proline was able to partially reverse the inhibition. The mechanism by which T 4C and A2C growth inhibition could be reversed by other amino acids (Table2) is not known. In higher plants (Reinhold and Kaplan, 1984) and algae (Reinhold and Kaplan, 1984; Sauer, 1984) amino acid transport systems have been reported which have affinities for all or groups of amino acids. In
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Chlorella vulgaris, growth inhibition by the methionine analog methionine sulfoximine could be reversed by glutamine because both glutamine and methionine sulfoximine competed for a common uptake system (Meins and Abrams, 1972). It is POSsible therefore that the mechanism by which some amino acids can reverse T 4C and A2C inhibition is by competition for uptake by a common transport system. In prokaryotes it has been reported that sensitivity to proline analogues increased in media of elevated osmolarity (Csonka, 1982; Sugiura and Kisumi, 1985). For example, in Salmonella typhimurium this was attributed to an additional proline permease which functions only in media of elevated osmolarity (Csonka, 1982) and served to accumulate higher levels of the inhibitors. At low salinity a proline producing strain of Serratia marcescans was completely resistant to 10mM A2C, T4C and DHP. However, in the presence of 500 mM NaCl, 1 mM A2C or DHP significantly inhibited growth (Sugiura and Kisumi, 1985). In contrast to previous reports with procaryotic organisms, the present work represents the first example of a reversal of proline analogue-mediated inhibition by high salinity. This reversal of inhibition by A2C is most likely to be due to the high intracellular proline pool in these cells. If the mechanism of inhibition by A2C in this alga is by incorporation into protein as has been found in other organisms (Fowden and Richmond, 1963; Troxler and Brown, 1974; Cella et aI., 1982), then the reversal of A2C growth inhibition by the high intracellular proline pool at high salinity suggests that this cytoplasmic pool may be available for protein synthesis. Acknowledgements Financial support was provided by the Natural Sciences and Engineering Research Council of Canada. References AHMAD, 1. and J. A. HELLEBUST: Osmoregulation in the extremely euryhaline marine micro-alga Chlorella autotrophica. Plant Physiol. 74, 1010-1015 (1984). BROWN, L. M.: Production of axenic cultures of algae by an osmotic method. Phycologia 21, 408-410 (1982 a). - Photosynthetic and growth responses to salinity in a marine isolate of Nannochloris baei/· laris (Chlorophyceae). J. Phycol. 18, 483 - 488 (1982 b). BROWN, L. M. and B. ELFMAN: Is autosporulation a feature of Nannochloris? Can. J. Bot. 61, 2647 -2657 (1983). BROWN, L. M. and J. A. HaLEBUST: Sorbitol and proline as intracellular osmotic solutes in the green alga Stichococcus bacillaris. Can. J. Bot. 56, 676-679 (1978). - - The contribution of organic solutes to osmotic balance in some green and eustigmatophyte algae. J. Phycol. 16, 265-270 (1980). CELLA, R., B. PARISI, and E. NIELSEN: Characterization of a carrot cell line resistant to azetidine2-carboxylic acid. Plant Sci. Lett. 24, 125-135 (1982). CSONKA, L. N.: A third L-proline permease in Salmonella typhimurium which functions in media of elevated osmotic strength. J. Bacteriol. 151, 1433-1443 (1982). ELTHON, T. E. and C. R. STEWART: Effects of the proline analog L-thiazolidine-4-carboxylic acid on proline metabolism. Plant Physiol. 74, 213-218 (1984).
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FOWDEN, L. and M. H. RICHMOND: Replacement of proline by azetidine-2-carboxylic acid during biosynthesis of protein. Biochim. Biophys. Acta 71, 459-461 (1963). HEWITT, E. J. and B. A. NOTTON: Inhibition by L-azetidine-2-carboxylic acid of induction of nitrate reductase in plants and its reversal by L-proline. Phytochemistry 6, 1329-1335 (1967). KUEH, J. S. H. and W. J. BRIGHT: Proline accumulation in a barley mutant resistant of trans-4hydroxyl-L-proline. Planta 153,166-171 (1981). - - Biochemical and genetical analysis of 3 proline-accumulating barley mutants. Plant Sci. Lett. 27, 233-241 (1982). KUEH, J. S. H., J. M. HILL, S. J. SMITH, and S. W. J. BRIGHT: Proline biosynthesis in a proline-accumulating barley mutant. Phytochemistry 23, 2207 -2210 (1984). LODATO, R. F., R. J. SMITH, D. L. VALLE, and K. CRANE: Mutant cell lines resistant to azetidine-2carboxylic acid: Alterations in the synthesis of proline from glutamic acid. J. Cell. Physiol. 119, 137 -143 (1984). MEINS, F. and M. A. ABRAMS: How methionine and glutamine prevent inhibition of growth by methionine sulfoximine. Biochim. Biophys. Acta 266, 307 - 311 (1972). MILER, P. M. and C. R. STEWART: Pyrroline-5-carboxylic acid reductase from soybean leaves. Phytochemistry 15, 1855-1857 (1976). NAKAMORI, S., H. MORIOKA, F. YOSHIMAGA, and S. YAMANAKA: Fermentative production of Lproline of DL-3,4-dehydroproline resistant mutants of L-glutamate producing bacteria. Agric. BioI. Chern. 46, 487 -491 (1982). NORRIS, R. D. and L. FOWDEN: Substrate discrimination by prolyl-tRNA synthetase from various higher plants. Phytochemistry 11, 2921-2935 (1972). PETERSON, P. J. and L. FOWDEN: Purification properties and comparative specificities of the enzyme prolyl-transfer ribonucleic acid synthetase from Phaseolus aureus and Polygonatum multiflorum. Biochem. J. 97, 112-124 (1965). REINHOLD, L. and A. KAPLAN: Membrane transport of sugars and amino acids. Ann. Rev. Plant. Physiol. 35,45-83 (1984). RiCCARDI, G., S. SORA, and O. CIFFERRI: Production of amino acids by analog-resistant mutants of the Cyanobacterium Spirulina platensis. J. Bacteriol. 147, 1002-1007 (1981). SAUER, N.: A general amino-acid permease is inducible in Chlorella vulgaris. Planta 161, 425431 (1984). SUGIURA, M. and M. KISUMI: Proline-hyperproducing strains of Seratia marcescens: Enhancement of proline analog-mediated growth inhibition by increasing osmotic stress. App!. Environ. Microbiol. 49, 782-786 (1985). TREICHEL, S., E. BRINCKMANN, B. SCHEITLER, and D. J. VON WILLERT: Occurrence and changes of proline content in plants in the southern Namib Desert in relation to increasing and decreasing drought. Planta 162, 236-242 (1984). TROXLER, R. F. and A. S. BROWN: Metabolism of L-azetidine-2-carboxylic acid in the alga Cyani· dium caldarium. Biochim. Biophys. Acta 366, 341-349 (1974). VAUGHAN, D. and E. CUSENS: Effects of hydroxyproline on the growth of excised root segments of Pisum sativum under aseptic conditions. Planta 112, 243 -252 (1973). VOETBERG, G. and C. R. STEWART: Steady state proline levels in salt-shocked barley leaves. Plant Physiol. 76, 567 - 570 (1984). WASMUTH, J. J. and C. T. CASKEY: Biochemical characterization of azetidine carboxylic acid-resistant Chinese hamster cells. Cell 8, 71-77 (1976). WIDHOLM, J. M.: Selection and characterization of cultured carrot and tobacco cells resistant to lysine, methionine and proline analogs. Can. J. Bot. 54, 1523-1529 (1976). YANCEY, P. H., M. E. CLARK, S. C. HAND, R. D. BOWLUS, and G. V. SOMERO: Living with water stress. Evolution of osmolyte systems. Science 217, 1214-1222 (1982).
]. Plant Physiol. Vol. 123. pp. 229- 239 (1986)