Planta
Planta 146, 629 633 (1979)
9 by Springer-Verlag 1979
Lysine Metabolism in a Barley Mutant Resistant to S(2-Aminoethyl)Cysteine Simon W.J. Bright, Leigh C. Featherstone, and Benjamin J. Miflin Biochemistry Department, Rothamsted Experimental Station, Harpenden, Herts. AL5 2JQ, U.K.
Abstract. Lysine and S(2-aminoethyl)cysteine (AEC) metabolism were investigated in n o r m a l barley (Hordeum vulgare L. cv. Bomi) and a h e m o z y g o u s recessive AEC-resistant m u t a n t (R906). F e e d b a c k regulation o f lysine and threonine synthesis f r o m [14C] acetate was unimpaired in plants o f the m u t a n t 3 d after germination. Seeds o f Bomi and R906 contained similar total a m o u n t s o f lysine, threonine, methionine and isoleucine. C o n c e n t r a t i o n s o f these a m i n o acids in the soluble fraction o f plants grown 6 d without A E C were also similar. The concentration o f A E C in R906 plants was less t h a n in the parent variety when b o t h were g r o w n in the presence o f 0.25 m M A E C for 6 d. The u p t a k e o f [3H]AEC and [3H]lysine by roots o f R906 was, respectively, 33% and 32% o f that by Bomi roots whereas the uptake of these c o m p o u n d s into the scutellum was the same in both the m u t a n t and its parent. The uptake o f [3H]leucine and its i n c o r p o r a t i o n into proteins was also the same in Bomi and R906 plants. These results suggest that a transport system specific for lysine and A E C but not leucine is altered or lost in roots o f the m u t a n t R906. A E C is i n c o r p o r a t e d into protein and this could be the reason for inhibition o f g r o w t h rather t h a n action as a false-feedback inhibitor o f lysine biosynthesis. Key words: Biochemical m u t a n t - F e e d b a c k regulation Hordeum - Lysine - M u t a n t S(2-aminoethyl)cysteine
Introduction Biochemically defined m u t a n t s o f higher plants have a potential use in studies o f plant metabolism, genetic
Abbreviations: AEC = S(2-aminoethyl)cysteine; THR = threonine
LYS = lysine;
manipulation and crop improvement. The lysine content o f the barley grain is p o o r and one possible way to improve it is to accumulate free lysine in the seed. S(2-aminoethyl)-L-cysteine (AEC) is a lysine analogue and has been used to select for lysine accumulation in bacteria (Sano and Shiio, 1970) and higher plant tissue cultures (Chaleff and Carlson, 1975; Widholm, 1976). We have selected a barley m u t a n t R906 resistant to A E C by screening mutagenised embryos for growth in the presence o f 0.25 m M A E C . Resistance is determined by a single recessive nuclear gene (aec-1) (Bright et al., 1979). We here report studies on the biochemical basis o f A E C inhibition in n o r m a l plants and the m e c h a n i s m o f resistance in R906.
Materials and Methods The isolation and genetic characterisation of the homozygous recessive R906 mutant of barley (Hordeum vulgare L. cv. Bomi) have been described (Bright et al., 1979). Embryos from mature seed were hand dissected and grown on the growth medium previously described with appropriate amino acid additions (Bright et al., 1978b). Amino Acid Analysis : Duplicate samples of plants grown 6 d +_0.25 mM AEC (mean fresh weights of Bomi plants were 125 and 118 mg without AEC and 24 and 27 mg with AEC; for R906 the corresponding values were 104, 70, 44, 43 rag) were freeze-dried, ground in a mortar and pestle in a little diethyl ether, washed three times in ether and soluble amino acids extracted from the remaining pellet by boiling three times for 5 min with 4 ml 70% (v/v) ethanol. The ethanol extract was evaporated to dryness, hydrolysed as previously described (Bright et al., 1978a) and amino acids analysed on a Technicon autoanalyser. When [3H] lysine was added to samples at the stage of ethanol extraction 77-83% was recovered in the final solutions. No correction for losses was made. Seeds of Bomi and R906 were milled to pass through a 0.7 mm sieve and the flour (100 rag) hydrolysed for amino acid analysis. N was determined by Kjeldahl analysis. Radioactive labelling: The [3H] AEC was obtained by catalytic exchange reaction of tritiated water with 1 g L-AEC at the Radiochemical Centre, Amersham, U.K. The crude product was purified
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630 by elution in 2M HC1 from Dowex-50W-X-8 H + form followed by paper chromatography (Whatman 3MM) in solvent 1 (see below) and elution in water. No other ninhydrin positive compounds were present in the AEC solution. The degree of racemisation of the L-AEC during tritiation was not determined. Other radioactive substances were obtained front the Radiochemical Centre. They were used at the following concentrations: L-[4,5-3H] lysine 12.53.107 ds -1 mmol 1, 3.13.104 ds 1 ml 1; L_[4,5_3H] leucine 1.85.108 ds -1 mmo1-1, 1.85-104 ds -1 ml i; [U_3H]AEC 2.32- 108 ds- 1 m m o l - 1, 5.81-104 d s - 1 m l - 1 ; sodium [2-~4C]acetate 2.07.109 ds -1 mmol-1, 6.48-104 ds 1 m l - t . For labelling studies plants were grown 2 d on agar growth medium and transferred to sterile 0.2 ml drops of liquid growth medium in petri dishes inside a glass-topped humid chamber for overnight preincubation. Uptake studies were performed by removing plants from their drops, blotting and transferring them to fresh 0.2 ml drops containing radioactive amino acids. After incubation in the humid chamber, plants were removed from the radioactive solutions, blotted, rinsed three times in 10 m M unlabelled amino acid solution in growth medium at 0 ~ C, blotted and frozen in liquid nitrogen. Soluble amino acids were obtained by three extractions in 1 ml methanol:chloroform : water (12 : 5 : 3 by vol) followed by separation into an aqueous layer (Bieleski and Turner, 1966). Protein pellets and dissected plant parts were solubilised in NCS tissue solubiliser (Hopkins and Williams Ltd.). Protease digestion was performed on extracts of Bomi plants incubated for 5 h with [U-3H]AEC. Soluble amino acids were extracted in cold 5% trichloracetic acid; 4plants were ground in 3 ml followed by three washes of 1 ml centrifugation. The pellet remaining was washed three times with 1 ml diethyl ether and incubated with protease (Pronase, Sigma type VI, 1 mg m l - 1) for 24 h at 37 ~ C in 3 ml Tris HC1 buffer, 50 m M pH 7.9, I m M CaClz. The reaction was terminated by addition of 3 ml cold 10% trichloracetic acid and the precipitate was collected by centrifugation, washed in ether and the protease digestion repeated once. For feeding [~4C]acetate, 11 plants were pooled per treatment. Plants were preincubated in the presence of the appropriate amino acids. Incubation was for 4 h in drops with amino acids and [t4C]acetate, by which time 91-94% of label had been taken up. Plants were harvested, blotted and freeze-dried. Soluble amino acids were extracted in methanol:chloroform:water as above. Asparagine and glutamine in the soluble fractions were deamidated by treatment with 5 ml 2M HC1 for 1 h at 95-100 ~ C. Soluble amino acids were then purified and protein pellets hydrolysed as previously described (Bright et al., 1978 a). Solvents used for chromatography were (1) butanol: acetic acid:water (12 : 3 : 5); (2) iso-propanol: formic acid: water (20 : 1 : 5) ; (3) butanol: acetone: diethylamine :triethylamine:water (10: 10:1 : 1 : 5); (4) pyridine:acetic acid:water (20:9.5:970) pH 5.0. Radioactivity in threonine was separated by two dimensional thin layer chromatography on cellulose in solvents 3 and 2 (Davies and Miflin, 1978). Radioactivity in lysine was separated by thin layer electrophoresis on cellulose in solvent 4 followed by chromatography in solvent 3. This removed a radioactive compound present in all AEC-treated samples which otherwise cochromatographed with lysine. Radioactivity in lysine and threonine was counted as in Davies and Miflin (1978). Tritium counts were corrected to disintegrations/min.
Results
The regulation of synthesis of lysine and threonine in vivo was examined by feeding [t4C]acetate to plants in the presence of lysine, threonine, lysine plus threonine or AEC and extracting the radioactively labelled lysine and threonine. The effects of the natu-
S.W.J. Bright et al.: Lysine Metabolism in Barley
A
8
Incorporation (% value with no amino acids)
1
)
LYS
THR
LYS THR
LYS
Treatment
THR
LYS THR
( 0 - 2 5 raM)
Fig. 1. The effect of lysine and threonine on the synthesis of lysine (A) and threonine (B) from [14C]acetate in Bomi (rn) and R906 ( i ) ; means of duplicate treatments except for Bomi, lys + thr
C 236 -
Incor poration (% value w i t h no
amino acids}
-100
5~ LYS
AEC Treatment
LYS
AEC
(0-25 raM)
II
LYS
AEC
Fig. 2. The effect of AEC and lysine on lysine synthesis from [14C]acetate in Bomi ([]) and R906 ( i ) . Means of duplicate treatments. Total: A; Protein fraction: B; Soluble fraction: C
ral amino acids on their own synthesis are shown in Fig. 1. Exogenous lysine inhibited accumulation of radioactivity in lysine and exogenous threonine inhibited incorporation into threonine. In this there was no difference between Bomi and R906. The effects o f lysine and AEC on incorporation of label into soluble and protein lysine were also studied. AEC at 0.25 m M inhibited the synthesis of lysine to the same extent as 0.25 m M lysine in Bomi plantlets whereas this was not so in the mutant (Fig. 2). The inhibition of lysine synthesis was only manifested in the protein fraction. The levels of free amino acids in the mutant and parent plants were compared by ethanol extraction of whole plants grown for 6 days+0.25 m M AEC (Table 1). It should be noted that the size of the plants in the control treatments was comparable but that Bomi plants on AEC-containing medium were considerably smaller than R906 ones. In the absence of AEC there was no appreciable difference in the concentrations of free aspartate-derived amino acids.
S.W.J. Bright et al. : Lysine Metabolism in Barley
631
Table 1. Freee amino acids in plants of Bomi and R906 grown 6 days• 0.25 mM AEC Amino acid (gmol/gFW)
250
Plants Bomi
2OO
R906
Medium -AEC
+AEC
-AEC
+AEC
0.83 0.86
1.23 1.17
0.78 0.82
1.03 1.05
-
0.81 0.82
-
0.54 0.51
Threonine
1.82 1.78
2.35 1.98
1.53 1.63
1.56 1.93
Methionine
0.76 0.24
0.50 0.39
0.23 0.22
0.27 0.30
Isoleucine
1.07 0.65
1.35 1.29
0.71 0.75
0.91 1.10
Lysine AEC
150 AEC uptake (dpm x 10"3/ 4 plants) 100
50
15
30 Time
Table 2. Total seed amino acids from hydrolysed flour of Bomi (17.98 mg N/g dw) and R906 (20.04mg N/g dw). Values are mean • std deviation of triplicate analyses Amino Acid (lamol/g dw)
Lysine Threonine Methionine Isoleucine
60 (rnin)
120
Fig. 3. Uptake of[3H]AEC (0.25 mM) into leaves ([], -), scutellum (~, A) and roots (9 o) of Bomi ([],z~,9 and R906 (m, A, e). Each point is the mean of triplicate treatments, 4 plants per treatment
Seed Bomi
R906
25.4_+ 1.3 29.6 _+0.8 12.5 • 2.7 32.2 + 0.7
27.1 _+4.0 36.1 _+4.6 16.3 • 6.8 38.6 _+8.2
200
150 Lysine uptake (dpm x I0-3/ 4 plants)
In the presence of AEC there were increases in all of the amino acids. The concentration of AEC in the mutant plants was however only 64% of that in the Bomi plants. Clearly, if there is no increase in free lysine in the vegetative parts of the plant then it is unlikely that there would be an increase in total seed lysine. This was confirmed by analysis of seed flour hydrolysates (Table 2). The decreased concentration of free AEC in the mutant plants grown in the presence of AEC suggested that the lesion in the mutant could be in the uptake or accumulation of AEC. Accordingly the uptake of [3H]AEC into roots, scutellum and leaves was measured over a 2 h period (Fig. 3). Over this period the mutant took up less AEC into the roots whilst passage of AEC into the scutellum was the same for both plants. The same pattern was obtained when uptake of [3H]lysine was examined (Fig. 4). In both cases the entry of amino acid into the leaves was very slow and it is not possible to draw any conclusions as to the relative rates of transfer to the leaves in the mutant and parent variety. Because of
10C
5O
15
30 Time
60 (min)
120
Fig. 4. Uptake of[3H]lysine (0.25 mM) into leaves ([], -), scutellum (A, A) and roots (9 e) of Bomi (n, zx, o) and R906 ( I , A, e). Each point is the mean of triplicate treatments, 4 plants per treatment
the unknown degree of isomerisation of the [3H]AEC being used it is not possible precisely to compare the rates of uptake of lysine and AEC. However, between 30 and 120 min R906 roots took up 77% less lysine and 78% less AEC than Bomi roots, indicat-
632
S.W.J. Bright et al. : Lysine Metabolism in Barley
c1 3 Leucine uptake
4 plants
1
Sample: d p m x 10 -3 (%)
Fraction
j
dprn x 10-5/
Table 3. Protease digestion of material insoluble in cold 5% w/v trichloracetic acid after feeding Bomi plants for 5 h with [U-gH]A]~C
Trichloracetic acid washes
E
F
1 2 3 4
Ether washes Protease digests
dpm • 10-5/
Pellet
B
983.0 (72.1) 89.2 (6.5) 24.3 (1.8) 6.4 (0.5)
933.7 (72.2) 71.8 (5.5) 17.9 (1.4) 4.6 (0.4)
0.2 1
2 AEC uptake
A
(0.0i)
231.5 (17.0)
0.4 (0.03) 237.3 (18.3)
15.2 (1.1)
15.9 (1.2)
13.6
12.2 (0.9)
(1.0)
4 Total
4 plants
1,363.4 (100)
1,293.8 (99.9)
2
lh
5h
lh
5h
lh
5h
Fig. 5. Uptake of [3H]leucine (A, B,C) from 0.1 mM solution in the presence of 0.25 mM AEC and of [~H]AEC (D, E, F) from 0.25 mM solution into plants of Bomi (n) and R906 (m) at 1 and 5h. Total uptake: A, D; Protein fraction: B, E; Soluble fraction: C, F. Each column is the mean of triplicate treatments (4 plants per treatment) except for R906 1 h + A E C which is in duplicate
ing that the same mechanism is operating in both cases. To test whether all amino acids are taken up at a reduced rate in the mutant, the uptake of leucine and AEC was compared. The amino acids taken up by whole plantlets were separated into soluble and insoluble fractions (Fig. 5). The uptake and incorporation of leucine into the insoluble fraction was unaffected in the mutant. AEC uptake and incorporation into the insoluble fraction were both reduced by approximately the same amount in the mutant. Evidence was sought that [3H]AEC is incorporated unchanged into protein. Bomi plants were analysed after incubation for 5 h with [3H]AEC (Table 3). 80.2% of the total label in the plants was extracted by four washes with cold 5% trichloracetic acid. Of the remaining label 94.8% was solubilised by 2 treatments with a general protease. The nature of the label in protein was investigated by extracting two further samples of Bomi plants (fed as above) five times with methanol : chloroform: water and hydrolysing the insoluble material in 6M HC1 at 120~ C for 21 h. The aqueous soluble fraction contained 72.9% of the label and the hydrolysate 27.1%. There was negligible labelling of other fractions. The aqueous soluble and protein hydrolysates were chromatographed in solvent 1 on Whatman No. 4 paper and the radioactivity assayed. In the protein hydrolysates an average of
95.5% of counts comigrated with A E C standards. This is little different from the 93.9% purity o f the [3H]AEC which was fed. In the soluble fraction an average 78.7% of label comigrated with AEC and a further 15.8% was found in two other areas.
Discussion The experiments above have concentrated on the problem of why the mutant R906 grows better in the presence of 0.25 mM AEC than its parent, Bomi. Thus all the labelling studies have used 0.25 m M amino acids in the normal medium in which it is known that the growth differences occur, Three differences have been noted between R906 and Bomi. (1) There is less free AEC in R906 plants grown 6 d in the presence of AEC. (2) In R906 there is less effect of AEC on the synthesis of lysine from [t4C]acetate. (3) R906 has decreased uptake of lysine and AEC into roots. Four areas of metabolism have been found to be unchanged in the mutant. (1) Lysine and threonine inhibit their own synthesis from [14C]acetate in a similar way to normal barley and that reported for wheat plants (Bright et al., 1978a). (2) Free amino acid concentrations in uninhibited plants are the same in Bomi and R906. (3) Total seed amino acids are comparable in Bomi and R906. (4) Leucine uptake is unaffected in R906. From these experiments we suggest that the primary lesion in the R906 mutant is a loss or alteration of the root uptake system specific for lysine and AEC but not leucine. This explains why mutant roots penetrate and grow in agar medium containing AEC whereas Bomi roots are inhibited on contact (Bright et al., 1979). Whilst the observed decrease in accumulation of AEC and lysine by mutant roots explains the AEC-
S.W.J. Bright et al. : Lysine Metabolism in Barley
resistance of the mutant it offers few clues as to the molecular basis of the difference. Whether the low net entry of ARC in the mutant roots is due to some different uptake system, export of newly transported AEC or an alteration in the efficiency of a single carrier system remains for further study. Amino acid uptake by roots of cucumber has been suggested to occur by a common carrier for all amino acids (Watson and Fowden, 1975) whereas our data would suggest that in barley roots there are at least two carriers, one for lysine and AEC and another for leucine. In the scutellum there is a further system for lysine and ARC different to that in the root. A model of lysine uptake by barley roots has recently been proposed in which there are five separate phases of uptake with different kinetic properties (Soldal and Nissen, 1978). They suggest also that lysine and arginine are transported by the same multiphasic mechanism. Thus barley roots could have separate systems for basic and neutral amino acids as in E. coli (Oxender, 1972). Separate uptake systems have been suggested for glycine and methionine in mustard roots (Wright, 1962). From our experiments we suggest that the inhibition of plant growth by ARC is caused by incorporation of the analogue into protein rather than by acting as a false-feedback inhibitor of lysine biosynthesis. Evidence in favour of this conclusion is that: (1) AEC is incorporated into protein (Table 3) and can be recovered unchanged after acid hydrolysis. (2) Of the total AEC taken up in 5 h by normal plants over half is in soluble AEC and the next largest amount of label is in protein-bound AEC. (3) Greater than equimolar amounts of lysine are required to relieve growth inhibition whereas only small amounts should be needed to relieve a starvation for lysine. In particular, 0.25 mM lysine is sufficient to inhibit lysine synthesis by 72% (Fig. 2) whereas this amount will not relieve growth inhibition by 0.3 mM AEC (Bright et al., 1979). (4) In Fig. 2 ARC has no inhibitory effect on the incorporation of label from [14C]acetate into soluble lysine. Inhibition is only observed in the protein fraction suggesting that AEC is competing with lysine for places incorporation into protein displacing it into the soluble pool which can build up and inhibit lysine synthesis. (5) In rat liver and E. coli AEC can be activated by aminoacyl-tRNA-synthetase preparations (DeMarco et al., 1976) and can compete with lysine for incorporation into protein in rabbit reticulocytes (Rabinowitz and Fisher, 1961). (6) Compared with lysine AEC is a relatively poor inhibitor of aspartate kinase from barley and wheat (Shewry and Miflin, 1977; Bright, et al., 1978a) and wheat dihydrodipicolinic acid synthase (Mazelis, M.M. personal communication). Resistance to AEC in plant tissue cultures has
633
been associated with increased amounts of free lysine (Widholm 1976, 1978) and with decreased incorporation into proteins (Negrutiu et al., 1978) but so far no plants have been regenerated from the resistant lines. This report is the first characterisation of AECresistance in whole plants in which both the biochemical and genetical studies are possible. This work was supported by grant number 00473 from the European Economic Community. We would like to thank Maureen Leggatt for performing amino acid analyses and Peter Shewry for Kjeldahl analysis.
References Bieleski, R.L., Turner, N.A. : Separation and estimation of amino acids in crude plant extracts by thin-layer electrophoresis and chromatography. Anal. Biochem. 17, 278 293 (1966) Bright, S.W.J., Norbury, P.B., Miflin, B.J. : Isolation of a recessive barley mutant resistant to S-(2-aminoethyl)-cysteine. Theor. Appl. Genet. 55, 1 4 (1979) Bright, S.W.J., Shewry( P.R., Miflin, B.J.: Aspartate kinase and the synthesis of aspartate-derived amino acids in wheat. Planta 139, 119-125 (1978a) Bright, S.W.J., Wood, E.A., Miflin, B.J. : The effect of aspartatederived amino acids (lysine, threonine, methionine) on the growth of excised embryos of wheat and barley. Planta 139, 113-117 (1978b) Chaleff, R.S., Carlson, P.S.: Higher plant cells as experimental organisms. In: Modification of the Information Content of Plant Cells. pp. 197-214, Markham, R., Davies, D.R., Hopwood, D.A., Horne, R.W., eds. New York: North-Holland 1975 Davies, H.M., Miflin, B.J. : Advantage of o-pthalaldehyde for visualising 14C-labelled amino acids on thin layer chromatograms and an improved method for their recovery. J. Chromatogr. 153, 284-286 (1978) DeMarco, C., Busiello, V., Girolamo, M. DI., Cavallini, D. : Selenalysine and protein synthesis. Biochim. Biophys. Acta 454, 298-308 (1976) Negrutiu, I., Jacobs, M., Cattoir, A. : Arabidopsis thaliana L. esp+ce modble en g+n+tique cellulair. Physiol. Veg. 16, 365 379 (1978) Oxender, D.L.: Membrane transport. Annu. Rev. Biochem. 41, 777-809 (1972) Rabinowitz, M., Fisher, J.M.: Formation of a ribosomal lesion in rabbit reticulocytes by the lysine antagonist, S-(fi-aminoethyl)cysteine. Biochem. Biophys. Res. Commun. 6, 449 451 (1961) Sano, K., Shiio, I. : Microbial production of L-lysine III: production by mutants resistant to S-(2-aminoethyl)-L-cysteine. J. Gen. Appl. Microbiol. 16, 373 391 (1970) Soldal, T., Nissen, P. : Multiphasic uptake of amino acids by barley roots. Physiol. Plant 43, 181-188 (1978) Watson, R., Fowden, L. : The uptake of phenylalanine and tyrosine by seedling root tips. Phytochemistry 14, 1181-1186 (1975) Widholm, J.M. : Selection and characterisation of cultured carrot and tobacco cells resistant to lysine, methionine and proline analogues. Can. J. Bot. 54, 1523-1529 (1976) Widholm, J.M. : Selection and characterisation of a Daucus carota L. cell line reistant to four amino acid analogues. J. Exp. Bot. 29, 1111-1116 (1978) Wright, D.E. : Amino acid uptake by plant roots. Arch. Biochem. Biophys. 97, 174-180 (1962)
Received 10 May; accepted 1 June 1979
Planta 146, 629 633 (1979)
9 by Springer-Verlag 1979
Lysine Metabolism in a Barley Mutant Resistant to S(2-Aminoethyl)Cysteine Simon W.J. Bright, Leigh C. Featherstone, and Benjamin J. Miflin Biochemistry Department, Rothamsted Experimental Station, Harpenden, Herts. AL5 2JQ, U.K.
Abstract. Lysine and S(2-aminoethyl)cysteine (AEC) metabolism were investigated in n o r m a l barley (Hordeum vulgare L. cv. Bomi) and a h e m o z y g o u s recessive AEC-resistant m u t a n t (R906). F e e d b a c k regulation o f lysine and threonine synthesis f r o m [14C] acetate was unimpaired in plants o f the m u t a n t 3 d after germination. Seeds o f Bomi and R906 contained similar total a m o u n t s o f lysine, threonine, methionine and isoleucine. C o n c e n t r a t i o n s o f these a m i n o acids in the soluble fraction o f plants grown 6 d without A E C were also similar. The concentration o f A E C in R906 plants was less t h a n in the parent variety when b o t h were g r o w n in the presence o f 0.25 m M A E C for 6 d. The u p t a k e o f [3H]AEC and [3H]lysine by roots o f R906 was, respectively, 33% and 32% o f that by Bomi roots whereas the uptake of these c o m p o u n d s into the scutellum was the same in both the m u t a n t and its parent. The uptake o f [3H]leucine and its i n c o r p o r a t i o n into proteins was also the same in Bomi and R906 plants. These results suggest that a transport system specific for lysine and A E C but not leucine is altered or lost in roots o f the m u t a n t R906. A E C is i n c o r p o r a t e d into protein and this could be the reason for inhibition o f g r o w t h rather t h a n action as a false-feedback inhibitor o f lysine biosynthesis. Key words: Biochemical m u t a n t - F e e d b a c k regulation Hordeum - Lysine - M u t a n t S(2-aminoethyl)cysteine
Introduction Biochemically defined m u t a n t s o f higher plants have a potential use in studies o f plant metabolism, genetic
Abbreviations: AEC = S(2-aminoethyl)cysteine; THR = threonine
LYS = lysine;
manipulation and crop improvement. The lysine content o f the barley grain is p o o r and one possible way to improve it is to accumulate free lysine in the seed. S(2-aminoethyl)-L-cysteine (AEC) is a lysine analogue and has been used to select for lysine accumulation in bacteria (Sano and Shiio, 1970) and higher plant tissue cultures (Chaleff and Carlson, 1975; Widholm, 1976). We have selected a barley m u t a n t R906 resistant to A E C by screening mutagenised embryos for growth in the presence o f 0.25 m M A E C . Resistance is determined by a single recessive nuclear gene (aec-1) (Bright et al., 1979). We here report studies on the biochemical basis o f A E C inhibition in n o r m a l plants and the m e c h a n i s m o f resistance in R906.
Materials and Methods The isolation and genetic characterisation of the homozygous recessive R906 mutant of barley (Hordeum vulgare L. cv. Bomi) have been described (Bright et al., 1979). Embryos from mature seed were hand dissected and grown on the growth medium previously described with appropriate amino acid additions (Bright et al., 1978b). Amino Acid Analysis : Duplicate samples of plants grown 6 d +_0.25 mM AEC (mean fresh weights of Bomi plants were 125 and 118 mg without AEC and 24 and 27 mg with AEC; for R906 the corresponding values were 104, 70, 44, 43 rag) were freeze-dried, ground in a mortar and pestle in a little diethyl ether, washed three times in ether and soluble amino acids extracted from the remaining pellet by boiling three times for 5 min with 4 ml 70% (v/v) ethanol. The ethanol extract was evaporated to dryness, hydrolysed as previously described (Bright et al., 1978a) and amino acids analysed on a Technicon autoanalyser. When [3H] lysine was added to samples at the stage of ethanol extraction 77-83% was recovered in the final solutions. No correction for losses was made. Seeds of Bomi and R906 were milled to pass through a 0.7 mm sieve and the flour (100 rag) hydrolysed for amino acid analysis. N was determined by Kjeldahl analysis. Radioactive labelling: The [3H] AEC was obtained by catalytic exchange reaction of tritiated water with 1 g L-AEC at the Radiochemical Centre, Amersham, U.K. The crude product was purified
0032-0935/79/0146/0629/$ 01.00
630 by elution in 2M HC1 from Dowex-50W-X-8 H + form followed by paper chromatography (Whatman 3MM) in solvent 1 (see below) and elution in water. No other ninhydrin positive compounds were present in the AEC solution. The degree of racemisation of the L-AEC during tritiation was not determined. Other radioactive substances were obtained front the Radiochemical Centre. They were used at the following concentrations: L-[4,5-3H] lysine 12.53.107 ds -1 mmol 1, 3.13.104 ds 1 ml 1; L_[4,5_3H] leucine 1.85.108 ds -1 mmo1-1, 1.85-104 ds -1 ml i; [U_3H]AEC 2.32- 108 ds- 1 m m o l - 1, 5.81-104 d s - 1 m l - 1 ; sodium [2-~4C]acetate 2.07.109 ds -1 mmol-1, 6.48-104 ds 1 m l - t . For labelling studies plants were grown 2 d on agar growth medium and transferred to sterile 0.2 ml drops of liquid growth medium in petri dishes inside a glass-topped humid chamber for overnight preincubation. Uptake studies were performed by removing plants from their drops, blotting and transferring them to fresh 0.2 ml drops containing radioactive amino acids. After incubation in the humid chamber, plants were removed from the radioactive solutions, blotted, rinsed three times in 10 m M unlabelled amino acid solution in growth medium at 0 ~ C, blotted and frozen in liquid nitrogen. Soluble amino acids were obtained by three extractions in 1 ml methanol:chloroform : water (12 : 5 : 3 by vol) followed by separation into an aqueous layer (Bieleski and Turner, 1966). Protein pellets and dissected plant parts were solubilised in NCS tissue solubiliser (Hopkins and Williams Ltd.). Protease digestion was performed on extracts of Bomi plants incubated for 5 h with [U-3H]AEC. Soluble amino acids were extracted in cold 5% trichloracetic acid; 4plants were ground in 3 ml followed by three washes of 1 ml centrifugation. The pellet remaining was washed three times with 1 ml diethyl ether and incubated with protease (Pronase, Sigma type VI, 1 mg m l - 1) for 24 h at 37 ~ C in 3 ml Tris HC1 buffer, 50 m M pH 7.9, I m M CaClz. The reaction was terminated by addition of 3 ml cold 10% trichloracetic acid and the precipitate was collected by centrifugation, washed in ether and the protease digestion repeated once. For feeding [~4C]acetate, 11 plants were pooled per treatment. Plants were preincubated in the presence of the appropriate amino acids. Incubation was for 4 h in drops with amino acids and [t4C]acetate, by which time 91-94% of label had been taken up. Plants were harvested, blotted and freeze-dried. Soluble amino acids were extracted in methanol:chloroform:water as above. Asparagine and glutamine in the soluble fractions were deamidated by treatment with 5 ml 2M HC1 for 1 h at 95-100 ~ C. Soluble amino acids were then purified and protein pellets hydrolysed as previously described (Bright et al., 1978 a). Solvents used for chromatography were (1) butanol: acetic acid:water (12 : 3 : 5); (2) iso-propanol: formic acid: water (20 : 1 : 5) ; (3) butanol: acetone: diethylamine :triethylamine:water (10: 10:1 : 1 : 5); (4) pyridine:acetic acid:water (20:9.5:970) pH 5.0. Radioactivity in threonine was separated by two dimensional thin layer chromatography on cellulose in solvents 3 and 2 (Davies and Miflin, 1978). Radioactivity in lysine was separated by thin layer electrophoresis on cellulose in solvent 4 followed by chromatography in solvent 3. This removed a radioactive compound present in all AEC-treated samples which otherwise cochromatographed with lysine. Radioactivity in lysine and threonine was counted as in Davies and Miflin (1978). Tritium counts were corrected to disintegrations/min.
Results
The regulation of synthesis of lysine and threonine in vivo was examined by feeding [t4C]acetate to plants in the presence of lysine, threonine, lysine plus threonine or AEC and extracting the radioactively labelled lysine and threonine. The effects of the natu-
S.W.J. Bright et al.: Lysine Metabolism in Barley
A
8
Incorporation (% value with no amino acids)
1
)
LYS
THR
LYS THR
LYS
Treatment
THR
LYS THR
( 0 - 2 5 raM)
Fig. 1. The effect of lysine and threonine on the synthesis of lysine (A) and threonine (B) from [14C]acetate in Bomi (rn) and R906 ( i ) ; means of duplicate treatments except for Bomi, lys + thr
C 236 -
Incor poration (% value w i t h no
amino acids}
-100
5~ LYS
AEC Treatment
LYS
AEC
(0-25 raM)
II
LYS
AEC
Fig. 2. The effect of AEC and lysine on lysine synthesis from [14C]acetate in Bomi ([]) and R906 ( i ) . Means of duplicate treatments. Total: A; Protein fraction: B; Soluble fraction: C
ral amino acids on their own synthesis are shown in Fig. 1. Exogenous lysine inhibited accumulation of radioactivity in lysine and exogenous threonine inhibited incorporation into threonine. In this there was no difference between Bomi and R906. The effects o f lysine and AEC on incorporation of label into soluble and protein lysine were also studied. AEC at 0.25 m M inhibited the synthesis of lysine to the same extent as 0.25 m M lysine in Bomi plantlets whereas this was not so in the mutant (Fig. 2). The inhibition of lysine synthesis was only manifested in the protein fraction. The levels of free amino acids in the mutant and parent plants were compared by ethanol extraction of whole plants grown for 6 days+0.25 m M AEC (Table 1). It should be noted that the size of the plants in the control treatments was comparable but that Bomi plants on AEC-containing medium were considerably smaller than R906 ones. In the absence of AEC there was no appreciable difference in the concentrations of free aspartate-derived amino acids.
S.W.J. Bright et al. : Lysine Metabolism in Barley
631
Table 1. Freee amino acids in plants of Bomi and R906 grown 6 days• 0.25 mM AEC Amino acid (gmol/gFW)
250
Plants Bomi
2OO
R906
Medium -AEC
+AEC
-AEC
+AEC
0.83 0.86
1.23 1.17
0.78 0.82
1.03 1.05
-
0.81 0.82
-
0.54 0.51
Threonine
1.82 1.78
2.35 1.98
1.53 1.63
1.56 1.93
Methionine
0.76 0.24
0.50 0.39
0.23 0.22
0.27 0.30
Isoleucine
1.07 0.65
1.35 1.29
0.71 0.75
0.91 1.10
Lysine AEC
150 AEC uptake (dpm x 10"3/ 4 plants) 100
50
15
30 Time
Table 2. Total seed amino acids from hydrolysed flour of Bomi (17.98 mg N/g dw) and R906 (20.04mg N/g dw). Values are mean • std deviation of triplicate analyses Amino Acid (lamol/g dw)
Lysine Threonine Methionine Isoleucine
60 (rnin)
120
Fig. 3. Uptake of[3H]AEC (0.25 mM) into leaves ([], -), scutellum (~, A) and roots (9 o) of Bomi ([],z~,9 and R906 (m, A, e). Each point is the mean of triplicate treatments, 4 plants per treatment
Seed Bomi
R906
25.4_+ 1.3 29.6 _+0.8 12.5 • 2.7 32.2 + 0.7
27.1 _+4.0 36.1 _+4.6 16.3 • 6.8 38.6 _+8.2
200
150 Lysine uptake (dpm x I0-3/ 4 plants)
In the presence of AEC there were increases in all of the amino acids. The concentration of AEC in the mutant plants was however only 64% of that in the Bomi plants. Clearly, if there is no increase in free lysine in the vegetative parts of the plant then it is unlikely that there would be an increase in total seed lysine. This was confirmed by analysis of seed flour hydrolysates (Table 2). The decreased concentration of free AEC in the mutant plants grown in the presence of AEC suggested that the lesion in the mutant could be in the uptake or accumulation of AEC. Accordingly the uptake of [3H]AEC into roots, scutellum and leaves was measured over a 2 h period (Fig. 3). Over this period the mutant took up less AEC into the roots whilst passage of AEC into the scutellum was the same for both plants. The same pattern was obtained when uptake of [3H]lysine was examined (Fig. 4). In both cases the entry of amino acid into the leaves was very slow and it is not possible to draw any conclusions as to the relative rates of transfer to the leaves in the mutant and parent variety. Because of
10C
5O
15
30 Time
60 (min)
120
Fig. 4. Uptake of[3H]lysine (0.25 mM) into leaves ([], -), scutellum (A, A) and roots (9 e) of Bomi (n, zx, o) and R906 ( I , A, e). Each point is the mean of triplicate treatments, 4 plants per treatment
the unknown degree of isomerisation of the [3H]AEC being used it is not possible precisely to compare the rates of uptake of lysine and AEC. However, between 30 and 120 min R906 roots took up 77% less lysine and 78% less AEC than Bomi roots, indicat-
632
S.W.J. Bright et al. : Lysine Metabolism in Barley
c1 3 Leucine uptake
4 plants
1
Sample: d p m x 10 -3 (%)
Fraction
j
dprn x 10-5/
Table 3. Protease digestion of material insoluble in cold 5% w/v trichloracetic acid after feeding Bomi plants for 5 h with [U-gH]A]~C
Trichloracetic acid washes
E
F
1 2 3 4
Ether washes Protease digests
dpm • 10-5/
Pellet
B
983.0 (72.1) 89.2 (6.5) 24.3 (1.8) 6.4 (0.5)
933.7 (72.2) 71.8 (5.5) 17.9 (1.4) 4.6 (0.4)
0.2 1
2 AEC uptake
A
(0.0i)
231.5 (17.0)
0.4 (0.03) 237.3 (18.3)
15.2 (1.1)
15.9 (1.2)
13.6
12.2 (0.9)
(1.0)
4 Total
4 plants
1,363.4 (100)
1,293.8 (99.9)
2
lh
5h
lh
5h
lh
5h
Fig. 5. Uptake of [3H]leucine (A, B,C) from 0.1 mM solution in the presence of 0.25 mM AEC and of [~H]AEC (D, E, F) from 0.25 mM solution into plants of Bomi (n) and R906 (m) at 1 and 5h. Total uptake: A, D; Protein fraction: B, E; Soluble fraction: C, F. Each column is the mean of triplicate treatments (4 plants per treatment) except for R906 1 h + A E C which is in duplicate
ing that the same mechanism is operating in both cases. To test whether all amino acids are taken up at a reduced rate in the mutant, the uptake of leucine and AEC was compared. The amino acids taken up by whole plantlets were separated into soluble and insoluble fractions (Fig. 5). The uptake and incorporation of leucine into the insoluble fraction was unaffected in the mutant. AEC uptake and incorporation into the insoluble fraction were both reduced by approximately the same amount in the mutant. Evidence was sought that [3H]AEC is incorporated unchanged into protein. Bomi plants were analysed after incubation for 5 h with [3H]AEC (Table 3). 80.2% of the total label in the plants was extracted by four washes with cold 5% trichloracetic acid. Of the remaining label 94.8% was solubilised by 2 treatments with a general protease. The nature of the label in protein was investigated by extracting two further samples of Bomi plants (fed as above) five times with methanol : chloroform: water and hydrolysing the insoluble material in 6M HC1 at 120~ C for 21 h. The aqueous soluble fraction contained 72.9% of the label and the hydrolysate 27.1%. There was negligible labelling of other fractions. The aqueous soluble and protein hydrolysates were chromatographed in solvent 1 on Whatman No. 4 paper and the radioactivity assayed. In the protein hydrolysates an average of
95.5% of counts comigrated with A E C standards. This is little different from the 93.9% purity o f the [3H]AEC which was fed. In the soluble fraction an average 78.7% of label comigrated with AEC and a further 15.8% was found in two other areas.
Discussion The experiments above have concentrated on the problem of why the mutant R906 grows better in the presence of 0.25 mM AEC than its parent, Bomi. Thus all the labelling studies have used 0.25 m M amino acids in the normal medium in which it is known that the growth differences occur, Three differences have been noted between R906 and Bomi. (1) There is less free AEC in R906 plants grown 6 d in the presence of AEC. (2) In R906 there is less effect of AEC on the synthesis of lysine from [t4C]acetate. (3) R906 has decreased uptake of lysine and AEC into roots. Four areas of metabolism have been found to be unchanged in the mutant. (1) Lysine and threonine inhibit their own synthesis from [14C]acetate in a similar way to normal barley and that reported for wheat plants (Bright et al., 1978a). (2) Free amino acid concentrations in uninhibited plants are the same in Bomi and R906. (3) Total seed amino acids are comparable in Bomi and R906. (4) Leucine uptake is unaffected in R906. From these experiments we suggest that the primary lesion in the R906 mutant is a loss or alteration of the root uptake system specific for lysine and AEC but not leucine. This explains why mutant roots penetrate and grow in agar medium containing AEC whereas Bomi roots are inhibited on contact (Bright et al., 1979). Whilst the observed decrease in accumulation of AEC and lysine by mutant roots explains the AEC-
S.W.J. Bright et al. : Lysine Metabolism in Barley
resistance of the mutant it offers few clues as to the molecular basis of the difference. Whether the low net entry of ARC in the mutant roots is due to some different uptake system, export of newly transported AEC or an alteration in the efficiency of a single carrier system remains for further study. Amino acid uptake by roots of cucumber has been suggested to occur by a common carrier for all amino acids (Watson and Fowden, 1975) whereas our data would suggest that in barley roots there are at least two carriers, one for lysine and AEC and another for leucine. In the scutellum there is a further system for lysine and ARC different to that in the root. A model of lysine uptake by barley roots has recently been proposed in which there are five separate phases of uptake with different kinetic properties (Soldal and Nissen, 1978). They suggest also that lysine and arginine are transported by the same multiphasic mechanism. Thus barley roots could have separate systems for basic and neutral amino acids as in E. coli (Oxender, 1972). Separate uptake systems have been suggested for glycine and methionine in mustard roots (Wright, 1962). From our experiments we suggest that the inhibition of plant growth by ARC is caused by incorporation of the analogue into protein rather than by acting as a false-feedback inhibitor of lysine biosynthesis. Evidence in favour of this conclusion is that: (1) AEC is incorporated into protein (Table 3) and can be recovered unchanged after acid hydrolysis. (2) Of the total AEC taken up in 5 h by normal plants over half is in soluble AEC and the next largest amount of label is in protein-bound AEC. (3) Greater than equimolar amounts of lysine are required to relieve growth inhibition whereas only small amounts should be needed to relieve a starvation for lysine. In particular, 0.25 mM lysine is sufficient to inhibit lysine synthesis by 72% (Fig. 2) whereas this amount will not relieve growth inhibition by 0.3 mM AEC (Bright et al., 1979). (4) In Fig. 2 ARC has no inhibitory effect on the incorporation of label from [14C]acetate into soluble lysine. Inhibition is only observed in the protein fraction suggesting that AEC is competing with lysine for places incorporation into protein displacing it into the soluble pool which can build up and inhibit lysine synthesis. (5) In rat liver and E. coli AEC can be activated by aminoacyl-tRNA-synthetase preparations (DeMarco et al., 1976) and can compete with lysine for incorporation into protein in rabbit reticulocytes (Rabinowitz and Fisher, 1961). (6) Compared with lysine AEC is a relatively poor inhibitor of aspartate kinase from barley and wheat (Shewry and Miflin, 1977; Bright, et al., 1978a) and wheat dihydrodipicolinic acid synthase (Mazelis, M.M. personal communication). Resistance to AEC in plant tissue cultures has
633
been associated with increased amounts of free lysine (Widholm 1976, 1978) and with decreased incorporation into proteins (Negrutiu et al., 1978) but so far no plants have been regenerated from the resistant lines. This report is the first characterisation of AECresistance in whole plants in which both the biochemical and genetical studies are possible. This work was supported by grant number 00473 from the European Economic Community. We would like to thank Maureen Leggatt for performing amino acid analyses and Peter Shewry for Kjeldahl analysis.
References Bieleski, R.L., Turner, N.A. : Separation and estimation of amino acids in crude plant extracts by thin-layer electrophoresis and chromatography. Anal. Biochem. 17, 278 293 (1966) Bright, S.W.J., Norbury, P.B., Miflin, B.J. : Isolation of a recessive barley mutant resistant to S-(2-aminoethyl)-cysteine. Theor. Appl. Genet. 55, 1 4 (1979) Bright, S.W.J., Shewry( P.R., Miflin, B.J.: Aspartate kinase and the synthesis of aspartate-derived amino acids in wheat. Planta 139, 119-125 (1978a) Bright, S.W.J., Wood, E.A., Miflin, B.J. : The effect of aspartatederived amino acids (lysine, threonine, methionine) on the growth of excised embryos of wheat and barley. Planta 139, 113-117 (1978b) Chaleff, R.S., Carlson, P.S.: Higher plant cells as experimental organisms. In: Modification of the Information Content of Plant Cells. pp. 197-214, Markham, R., Davies, D.R., Hopwood, D.A., Horne, R.W., eds. New York: North-Holland 1975 Davies, H.M., Miflin, B.J. : Advantage of o-pthalaldehyde for visualising 14C-labelled amino acids on thin layer chromatograms and an improved method for their recovery. J. Chromatogr. 153, 284-286 (1978) DeMarco, C., Busiello, V., Girolamo, M. DI., Cavallini, D. : Selenalysine and protein synthesis. Biochim. Biophys. Acta 454, 298-308 (1976) Negrutiu, I., Jacobs, M., Cattoir, A. : Arabidopsis thaliana L. esp+ce modble en g+n+tique cellulair. Physiol. Veg. 16, 365 379 (1978) Oxender, D.L.: Membrane transport. Annu. Rev. Biochem. 41, 777-809 (1972) Rabinowitz, M., Fisher, J.M.: Formation of a ribosomal lesion in rabbit reticulocytes by the lysine antagonist, S-(fi-aminoethyl)cysteine. Biochem. Biophys. Res. Commun. 6, 449 451 (1961) Sano, K., Shiio, I. : Microbial production of L-lysine III: production by mutants resistant to S-(2-aminoethyl)-L-cysteine. J. Gen. Appl. Microbiol. 16, 373 391 (1970) Soldal, T., Nissen, P. : Multiphasic uptake of amino acids by barley roots. Physiol. Plant 43, 181-188 (1978) Watson, R., Fowden, L. : The uptake of phenylalanine and tyrosine by seedling root tips. Phytochemistry 14, 1181-1186 (1975) Widholm, J.M. : Selection and characterisation of cultured carrot and tobacco cells resistant to lysine, methionine and proline analogues. Can. J. Bot. 54, 1523-1529 (1976) Widholm, J.M. : Selection and characterisation of a Daucus carota L. cell line reistant to four amino acid analogues. J. Exp. Bot. 29, 1111-1116 (1978) Wright, D.E. : Amino acid uptake by plant roots. Arch. Biochem. Biophys. 97, 174-180 (1962)
Received 10 May; accepted 1 June 1979
