Threonine Accumulation in a Mutant of Arabidopsis thaliana (L.) Heynh. with an Altered Aspartate Kinase

f. Plant Pbysiol. Vol. 146. pp. 249-257 (1995)

Threonine Accumulation in a Mutant of Arabidopsis thaliana (L.) Heynh. with an Altered Aspartate Kinase BETTY HEREMANS

and MICHEL JACOBS

Vrije Universiteit Brussel, Institute for Molecular Biology and Biotechnology, Laboratory of Plant Genetics, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium Received September 20, 1994 ·Accepted January 5, 1995

Summary

After mutagenesis, M 2 seedlings of Arabidopsis thaliana were grown on a selective medium containing toxic concentrations of lysine and threonine. One of the LT-resistant mutants (RLT 40) was studied at the biochemical and genetical levels. A six-fold increase in free threonine content was found in 8-day-old mutant plantlets compared with the wild type. The total amino acid content of the mutant was also remarkably increased, essentially due to increased levels of aspartate, threonine, methionine, isoleucine and lysine. As one of the possible reasons for the LT -resistance resides in a change of the regulatory properties of the first enzyme of the aspartate pathway, aspartate kinase (EC 2.7.2.4), the feedback-inhibition pattern of aspartate kinase was examined in the mutant and the wild type. In Arabidopsis, after ion-exchange chromatography of whole plant extracts, three peaks of activity were detected corresponding respectively to a threonine-sensitive isoform, to a lysine-sensitive form and a form insensitive to both inhibitors. The threonine accumulation in RLT 40 could be related to a partial insensitivity of the lysinesensitive form of aspartate kinase. Genetical analysis showed that the resistance gene behaved as a dominant, monogenic nuclear trait. Linkage analysis, performed with a multiple marker line, indicated that the mutation is located on chromosome 2, 36.0cM from the er locus and 19.8 eM from the py locus on chromosome 2. This partially lysine-insensitive mutant of aspartate kinase offers ways to a map-based approach for cloning the gene coding for the corresponding isoform in plants.

Key words: Arabidopsis thaliana (L.) Heynh.; aspartate kinase; LT-resistance; threonine overproduction. Abbreviations: AEC == S-2-aminoethyl-L-cysteine; AK = aspartate kinase; EMS = ethylmethane sulphonate; FW = fresh weight; FPLC = fast protein liquid chromatography; LT = lysine plus threonine; SAM = S-adenosylmethionine. Introduction

In higher plants the amino acids lysine, threonine, methionine and isoleucine are synthesized from aspartate by a common branched pathway that is subjected to regulation by the end products. Both lysine and threonine exert regulating effects on aspartate kinase (EC 2.7.2.4), the first enzyme in the aspartate family amino acid pathway (Bryan, 1980). Also the first enzyme in the branched rate leading to lysine, dihydrodipicolinate synthase (EC 4.2.1.52), is strictly controlled by lysine, while the branch of the pathway leading to threonine, © 1995 by Gustav Fischer Verlag, Stuttgart

isoleucine and methionine is essentially controlled by threonine at the level of homoserine dehydrogenase (EC 1.1.1.3). The first enzyme of the aspartate pathway, aspartate kinase, catalyses the conversion of aspartate into ,3-aspartyl phosphate: Mgl+ aspartate + ATP-+ {j-aspartyl phosphate + ADP.

Aspartate kinase has been detected in many plant species (Azevedo et al., 1992; Frankard et al., 1991; Kochhar et al.,

250

BEITY HEREMANs and MrcHEL JAcoBs

1986; Lea et al., 1979; Piryns et al., 1988; Rognes et al., 1983; Wilson et al., 1991; Wong and Dennis, 1973; Wong and Dennis, 1973). This report describes the isolation and characterization of a LT-resistant mutant in Arabidopsis characterized by a threonine overproduction. Aspartate kinase, the regulatory properties of which are affected in the mutant, has been partially purified using anion-exchange chromatography and its biochemical properties analysed in the wild type as well as in the mutant. In this study Arabidopsis is chosen as model system. Besides its advantages for plant molecular genetics, such as a short life cycle, small size and small genome size, the small seeds contain only a small nutrient reserve and can be considered as isolated embryos. Therefore, selections to various amino acid analogues and inhibitory amino acid combinations in seeds allow large numbers of seedlings to be screened and a broad spectrum of mutants to be obtained (Heremans andJabobs, 1994).

Materials and Methods

Isolation ofthe mutant Arabidopsis thaliana (var. Bensheim) seeds were treated with 1% ethylmethane sulphonate (EMS) for 3 h and grown in the greenhouse. M2 seeds were screened for their resistance to inhibitory concentrations of lysine plus threonine. The selection concentration used was 1 mM lysine, 1 mM threonine and 0.5 mM arginine, all added to Feenstra-medium (Oostindier-Braaksma and Feenstra, 1973) after sterile filtration (Cattoir-Reynaerts et al., 1981). Arginine was added to the selective medium to prevent inhibition by lysine, unrelated to regulatory effects on enzymes of the aspartate biosynthetic pathway (Cattoir-Reynaerts et al., 1981). After 1 week, resistant plants were transferred to pot soil. Their progenies were tested for resistance by sowing seeds on the similar solid selective medium. Seeds were sterilized in saturated calcium hypochloride (w/v) for 20 min and washed 3 x 10 min with sterile water. Growth ofplant materialfor biochemical analysis Plants were grown under sterile conditions in 250 mL Erlenmeyer flasks in liquid Feenstra-medium (Oostindier-Braaksma and Feenstra, 1973). Seeds were sterilized by soaking them for 10 to 15 min in sterile tubes containing an acidified saturated calcium hypochloride solution (w/v) (0.5 mL of 0.7M acetic acid per 25 mL saturated calcium hypochloride). Then they were washed twice with sterile water and once with liquid Feenstra-medium for 10 min. Finally, the seeds were transferred to Erlenmeyer flasks containing approximately 150 mL medium. The medium was changed every 10 days to prevent exhaustion. These submerged cultures were grown on a shaker in a culture room at 23 °C and in a 16h light/8 h dark light cycle (Philips Hg-1 lamps 400W ·m-2).

Amino acid analysis Free amino acids. Between 100 and 200 mg of whole 8-day-old plantlets was ground and homogenized in a mortar with a mixture of methanol : chloroform :water (12 : 5 : 2) according to the slightly modified method of Bieleski and Turner (1966). At each of the three successive extractions, the homogenate was centrifuged (5 min 10,000 x g), and the supernatant pooled. Chlorophyll was removed by adding two parts of chloroform and one part of water to the extracts. The aqueous, amino acid-containing, upper layer was iso-

lated and evaporated to dryness. The residue was then resuspended in 1 mL water and 300 J.LL 2 N HCl, and placed under vacuum at 110 °C to hydrolyse asparagine and glutamine to their respectively acidic forms. After evaporation at 85 °C, the residue was resuspended in 1 mL sample buffer (0.1 M Na-citrate, pH 2.2) and analysed on BTC 2710 resin using a five buffer system and ninhydrin detection. Total amino acids (free + protein-bound). Ground plant material (50 -100 mg) in 1 N HCl was hydrolyzed in a sealed glass tube under vacuum at 110 °C for 24 h. The hydrolysate was then evaporated at 85 °C, resuspended in 1 mL sample buffer and analysed as described above.

Partial purification ofaspartate kinase All procedures were carried out at 4 °C.

Enzyme extraction. Eight- or 21-day-old plantlets were harvested,

washed with distilled water and the excess of water removed by dipping on filter paper. The plant material was extracted in a mortar by grinding with sand and 2mL extraction buffer (100mM K-phosphate, 1mM Na2"EDTA, 20% glycerol (v/v), 10mM diethyldithiocarbamate, 1 mM lysine, 1 mM threonine and 10 mM {3mercaptoethanol, pH 8.0) per g plant material. Lysine, threonine and {3-mercaptoethanol were added to the buffer just before use. In a following step, the extract was filtered through two layers of miracloth and centrifuged in a Sorvall SS-34 rotor at 6,315 x g for 10 min to remove cell debris. The proteins in the supernatant were precipitated by adding solid ammonium sulphate to 60% saturation and collected by centrifugation (6,315 x g for 10 min). The pellet was resuspended in 0.2 mL dialysis buffer [50 mM K-phosphate, 1 mM Na2-EDTA, 20% glycerol (v/v), 5mM {3-mercaptoethanol (added just before use), pH 7.5] per g plant material and passed through a Sephadex G-25 column (equilibrated in the same buffer) to remove ammonium sulphate and low molecular weight compounds. This enzyme preparation without further purification was used for determination of the catalytic properties of the enzyme, the stability and the inhibition pattern. When the enzyme was dissolved in the 50mM potassium-phosphate buffer, pH 7.5, it kept approximately 91% of its original activity after 48 h at 4 °C; 67% and 58% of the original activity was maintained respectively after 6 and 10 days at this temperature. Anion-exchange chromatography. For the separation of the different isoforms of aspartate kinase another extraction buffer (2 mL buffer per gram plant material) was used: 25 mM K-phosphate, 2mM Na2-EDTA, 2mM MgCl, 15% glycerol (v/v), 1mM lysine, 1 mM threonine and 0.2% {3-mercaptoethanol (v/v), pH 7.5. After protein precipitation and redissolving the extract in 0.2 mL per g plant material of the same buffer but without lysine and threonine, the extract was desalted on a Sephadex G-25 column and the green fractions containing aspartate kinase activity were pooled. Then 0.5 mL was loaded onto a Pharmacia Fast Protein Liquid Chromatography (FPLC) Mono Q HR 5/5 column (1 mL) equilibrated with the same buffer, without lysine and threonine. The column was developed at a flow rate of 0.5 mL/min and aspartate kinase was eluted with a 15mL gradient (50-450mM KCl) into 0.5mL fractions. These fractions were assayed for aspartate kinase activity. Gel filtration chromatography. Determinations of molecular weights were performed on a Pharmacia FPLC system. For gel filtration experiments on Superose 12, a column of 1.0 em x 28.2 em was used at a flow rate of 0.4 mL. The running buffer was 50 mM K-phosphate, 150 mM KCl, 1 mM dithiotreitol, and 10% ethanedial (v/v), pH 7.5, and samples of 0.5 mL were injected. Fractions (0.5 mL) were collected and assayed for aspartate kinase activity. The void volume (6.1 mL) was determined with Dextran blue 2000. The Superose 12 column was calibrated with several standard pro-

Threonine accumulation in aArabidopsis mutant teins: ribonuclease (13,700), ovalbumin (43,000), albumin (67,000), aldolase (158,000) and catalase (232,000). Assayfor aspartate kinase activity

Aspartate kinase activity from Arabidopsis extracts was assayed by the hydroxamate method of Black and Wright (1955). AK required the presence of a divalent cation for its activity; the highest activity was reached in the presence of Mgl+, while the activity was reduced to 29% in the absence of any divalent cation. Therefore, the reaction mixture consisted of 10mM ATP, 10mM MgCh, 50 mM K-aspartate (adjusted to pH 7.0 with KOH), 500 mM hydroxylamine (adjusted to pH 7.5 with KOH), enzyme extract and water up to a final volume of 0.6 mL. Following incubation at 30 °C for 60 min, the reaction was stopped by adding 0.5 mL of the FeC1 3-colour reagent (0.37M FeCh, 3.3% TCA (w/v), 0.7M HCl). The mixture was centrifuged for 5 min at 10,000 x g and the absorbance of the supernatant was measured at 505 nm. Blank reaction mixtures containing all the components except aspartate were always included as controls. Specific activity was expressed as nmol flaspartyl hydroxamate formed per min and per mg protein. The Km-values for aspartate and ATP, determined by this method, were 5.24 and 1.26 mM, respectively. Protein estimation

Protein concentration was determined using the method of Bradford, with a solution of bovine serum albumin as reference (Bradford, 1976). Genetical analysis

To assign the mutation of RLT 40 to one of the five chromosomes of Arabidopsis the mutant was crossed to different mapping lines. The two used Wageningen tester lines, which were obtained from M. Koornneef (Wageningen) and M. Anderson (Nottingham Arabidopsis Stock Centre), were descendent from the Landsberg erecta line of Arabidopsis. These lines included W100 (an, apl, er, py, hy2, gll, bp, cer2, tt3) and W130 (er, py, hy3). To study linkage repulsion phase crosses were performed between the Wageningen lines (female parent) and the threonine overproducer RLT 40 (male parent). The resulting Ft progeny were allowed to self-pollinate and the phenotypes of F2 individuals derived from single F1 plants were determined on the basis of the description of the Wageningen lines (Koornneef, 1990). The resistance to LT-medium was determined in F3 individuals, after self-pollination of the F2 plants, by sowing seeds on LT-selective medium. The recombination values between the various loci were assessed with the method of maximum likelihood (Serra, 1965). The estimates of recombination percentages were corrected for double crossovers by the Kosambi mapping function, converting them to map distances (D) in centiMorgans (eM) (Koornneef, 1990). The map positions of the phenotypic markers were obtained from the integrated genetic/RFLP linkage map of Arabidopsis thaliana (Hauge et al., 1993). Results

Isolation ofa LT-resistant mutant For isolation, 640,000 M 2 seedlings were subjected to iTselection and after repetition of the same test on M 3 progenies, 56 offsprings could be clearly identified as resistant (Heremans and Jacobs, 1994). These plants were either homogenously resistant or segregated into resistant and sensi-

251

tive plants. RLT 40 was one of the isolated mutants on LTmedium that segregated into a 3 : 1 resistant-sensitive ratio. Phenotypically, one could not distinguish the mutant from the wild type and the mutant showed a normal fertility. Resistant plants were further propagated and homozygous progenies were used for biochemical analysis.

Amino acid content The free amino acid content of both RLT 40 and wild type was compared in 8-day-old plantlets grown on basal medium. A 6-fold increase in soluble threonine content was found: 174nmoles/g FW in the wild type compared with 1,057nmoles/g FW in RLT 40 (Table 1). However, threonine was not the only amino acid of the aspartate pathway affected. Also, though to a lesser extent, an increase in free lysine content occurred (1.5 fold). In contrast, the free aspartate pool was reduced by approximately 40% (563 nmoles/g FW in the mutant against 1,479nmoles/g FW in the wild type) and the total content of free amino acids reached only 60% of that measured in the wild type. This clear decrease in total soluble amino acid concentration was for the greatest part due to the lower level of free aspartate and glutamate, although the content of the other amino acids was also reduced. Nevertheless, the lower free aspartate pool did not seem to limit the production of either amino acid derived from it. The total amino acid content (free + protein-bound) of the mutant was remarkably increased by circa 40% (Table 1). This was partially due to the higher content of aspartate (23%), threonine (175%), methionine (96%), isoleucine (87%) and lysine (30%). This increase in the total amino acid pool only resulted from a higher level of protein-bound amino acids, since the free amino acid pool of RLT 40, which is reduced compared with the wild type, represented only approximately 4% of the total amino acid content. We have no explanation for the remarkable augmentation in protein-bound amino acids.

Enzymatic properties One of the possible reasons for the LT -resistance could reside in a change of the regulatory properties of aspartate kinase (AK). Therefore, the inhibition pattern of AK was determined in the mutant, as well as in the wild type. The wild type enzyme was strongly inhibited by lysine and to a much lesser extent by threonine (Fig. 1). The IC50 value for the inhibition by lysine was 0.75 mM and a maximal inhibition was obtained with 5 mM lysine, where a plateau was reached. Only 15% of the enzyme activity appeared to be sensitive to threonine (5 mM). When both amino acids were added together an additive inhibition of the AK-activity was observed, while 15 to 20% of the enzyme remained insensitive to lysine plus threonine, suggesting the existence of two isozymes, one lysine-sensitive, the other threonine-sensitive, and possibly a third isoform, insensitive to feedback control by both lysine and threonine. However, the inhibition pattern of AK determined in the mutant RLT 40 (Fig. 2) clearly differed from the one estab-

252

BETIY HEREMANS

and MicHEL jACOBS

Table 1: Free and total (free + protein-bound) amino acid pool of 8-day-old seedlings in wild type and RLT 40; the results are expressed in absolute (mean ± standard error Om for n replicates) and in relative values. Amino acid

Free amino acid pool nmol/gFW

Aspartate Threonine Serine Glutamate Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Ornithine

1479 ± 76 174± 22 600 ± 80 4806 ± 151 170 ± 23 1109 ± 103 1064 ± 86 45 ± 11 121 ± 11 59± 10 42± 5 58± 5 28 ± 8 56± 6 324 ± 15 38 ± 3 240 ± 50 39 ± 6

Total

Total amino acid pool

RLT 40 (n=7)

Wild type (n -7) %

nmol/gFW

%

14.2 1.7 5.7 46.0 1.6 10.6 10.2 0.4 1.2 0.6 0.4 0.6 0.3 0.5 3.1 0.4 2.3 0.4

563 ± 1057 ± 678 ± 2246 ± 218 ± 392 ± 356 ± 19 ± 61± 21 ± 38 ± 133 ± 0± 25 ± 185 ± 56± 58± 0±

9.2 17.3 11.1 36.8 3.6 6.4 5.8 0.3 1.0 0.3 0.6 2.2 0.0 0.4 3.0 0.9 0.9 0.0

40 46 29 98 9 55 17 5 5 1 6 8 0 3 2 4 10 0

6107 ± 210

10453 ± 277

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nmol/gFW 12576 ± 5410 ± 6215 ± 17809 ± 7151 ± 11508 ± 11347 ± 242 ± 7346 ± 729 ± 5087 ± 8543 ± 2483 ± 4702 ± 2244 ± 7287 ± 5006 ± 293 ±

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RLT 40 (n=7) nmol/gFW 15444 ± 789 14898 ± 901 8337 ± 381 26195 ± 1621 8441 ± 371 15002 ± 728 15061 ± 389 320 ± 84 10778 ± 298 1427 ± 47 7996 ± 254 12972 ± 413 3919 ± 151 6527 ± 292 4396 ± 136 9523 ± 457 5016 ± 1123 0± 0

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lished for the wild type (Fig. 1). While the inhibition profile due to threonine remained unchanged, the feedback inhibition exerted by lysine {tested up to a concentration of 20 mM) on AK-activity did not exceed 48 %, while in the wild type 68% of the global AK-activity was sensitive to such a lysine concentration. It was also established that no significant change affects the specific AK-activity of RLT 40: the mutant enzyme showed an activity of 6.1 nmoles/mg prot. min against 6.4 nmoles/mg prot. min in the wild type. From this it could be deduced that the mutant displayed an AK approximately 70 % desensitized to feedback inhibition by lysine. When in Arabidopsis the inhibition of AK by lysine was tested in the presence of S-adenosylmethionine (SAM), a synergistic inhibitory effect was observed as already reported by Rognes et al. (1983), Relton et al. (1988) and Frankard et al. (1991). In the presence of 0.2mM SAM the enzyme sensitivity towards lysine inhibition was greatly enhanced: a 5-fold drop in the IC5o value from 0.75 mM to 150 JJM was found (Fig. 3). However, if SAM alone was present in a concentration up to 0.4 mM, SAM had little or no effect on AK-activity, but at higher concentrations (up to 2 mM) SAM strikingly stimulated the enzyme activity (Fig. 3). The apparent effector had a purity of 91% {tested by high pressure liquid chromatography). AK is known to be susceptible to heat inactivation (Relton et al., 1988). When enzyme preparations were incubated for 10 min at a range of temperatures from 5 to 60 °C, immediately centrifuged {10,000 X g for 10 min), cooled on ice and used for enzyme activity determination, the obtained heat inactivation curve revealed that AK lost 55 % of its original activity between 40 and 45 °C (Fig. 4). After heating at 60 °C, still 36% of the activity remained. Apparently, the enzyme was not yet completely denatured at this temper-

Threonine accumulation in aArabidopsis mutant ature. When considering the inhibition by lysine and threonine exerted on AK after heat inactivation, it became clear that the lysine-sensitive fraction presented a higher heat susceptibility than the threonine-sensitive fraction: the lysinesensitive fraction being already partly denatured at 30 °C, while the threonine-sensitive fraction was only affected at 45 °C. In the presence of both effectors, enzyme activity was still measured, even after heat treatment at 60 °C. These data may also be an indication for the existence of three isoforms of AK in A rabidopsis. Indeed, AK was reproducibly separated into three peaks of activity eluting from a FPLC Mono Q HR 5/5 anion-exchange column (Fig. 5). Peak I eluted from the column by 150 mM KCl; it was inhibited 20% by 10 mM threonine and less than 10% by 10 mM lysine and represented 54% of the global AK-activity (Table 2). Peak II was a lysine-sensitive form eluted by 250 mM KCl: 55% of the activity was inhibited by lysine and only less than 5% by threonine. Peak II contributed 34% to the total AK-activity. A third peak eluted at a higher concentration of KCl {340 mM) was less than 5 % inhibited by lysine as well as by threonine. Peak ill was responsible for 13% of the entire enzyme activity. Finally, the enzyme of RLT 40 also separated into 3 different 7.0

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Fig. 3: Effect of increasing SAM and SAM plus lysine concentrations on aspartate kinase activity. Specific enzyme activity of 8-dayold plants was assayed as described in «Materials and Methods»; results are represented as mean ± standard error Urn. peaks by FPLC (Fig. 6). AK I showed a similar sensitivity to threonine as in the wild type: AK I from RLT 40 was 17% inhibited by threonine, against 20% in the wild type (Table 2). However, up to 80% of the activity of AK II remained in the presence of lysine, while in the wild type only 45% of the enzyme activity was detected. Therefore, a marked decrease in the lysine-sensitivity was noted in this peak for the mutant compared with the wild type. The third peak, as in the wild type, appeared insensitive to lysine or threonine. As in the wild type, AK I represented more than 50% of the global activity in the mutant, and the contributions of AK II and ill to total enzyme activity were comparable with those found in the wild type. The molecular weight of the isoforms of AK was determined by Superose 12 gel filtration. The threonine-sensitive enzyme had a Mr of 183,014 ± 1,500, while the Mr of the lysine-sensitive form was estimated to be 238,071 ± 10,696. For the insensitive isozyme the Mr was not established.

Genetical analysis In the progeny of the cross between the mutant and W100, a segregation of 3 LT-resistant to 1 LT-sensitive plant was observed: among 163 F2 plants tested 123 plants were found

254

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and MicHEL jAcoBs

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Fig. 4: Effect of heat treatment on aspartate kinase activity associated with the presence of inhibiting concentrations of lysine and threonine (10mM). Specific enzyme activity of 21-day-old plants was assayed as described in «Materials and Methods»; results are represented as mean ± standard error Um.

Fig. 5: Elution profile of wild type aspartate kinase from an FPLC mono Q anion-exchange column (50-450 mM KCl), and sensitivity of the fractions in the presence of inhibitory concentrations of lysine and threonine (10mM).

to be LT-resistant (~ = 0.033, 0.90 < p < 0.80). Therefore, the resistance of the mutant can be ascribed to one single gene with a dominant behaviour. Linkage analysis performed in the F2 and F3 offspring from a cross between W100 and RL T 40 revealed that er and py markers on chromosome 2 displayed only a low recombination frequency with the ak2 mutation, indicating linkage with these two

morphological markers of chromosome 2 (Table 3). In a next step the mutant was crossed to W130, a Wageningen line containing three other morphological markers of chromosome 2. Analysis of the F2 progeny indicated that the ak2 and py loci were closely linked and that ak2 and hy3 loci behaved as unlinked {Table 3). Calculated map distances of the ak2 locus from the markers on chromosome 2 indicated that

Table 2: Characteristics of the 3 AK-isoforms fractionated by FPLC Mono Q anion-exchange chromatography with a KCl-gradient (50450 mM KCl) in the wild type and the mutant RLT 40. Results of specific AK-activity in the presence of lysine and threonine are expressed in absolute (nmol/mg ·min) and in relative values(%). Specific AK-activity nmol/mg ·min

AK-form No inhibitor

Relative fraction

10mM lysine

% inhibition

10mM threonine

% inhibition

Wild type

Peak 1: 150 mM KCl Peak 2: 250 mM KCl Peak 3: 337 mM KCl

991 629 232 1852

54% 34% 13%

897 281 225

9% 55% 3%

792 605 211

20% 4% 9%

RLT40

Peak 1: 163 mM KCl Peak 2: 257 mM KCl Peak 3: 337 mM KCl

842 523 229 1594

53% 33% 14%

781 421 224

7% 20% 2%

698 505 223

17% 3% 3%

Threonine accumulation in aArabidopsis mutant

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Fig. 6: Elution profile of mutant aspartate kinase from an FPLC mono Q anion-exchange column (50-450mM KCl), and sensitivity of the fractions in the presence of inhibitory concentrations of lysine and threonin~ (10mM). Table 3: Recombination frequencies between the LT-resistance (R-) of RLT 40 and some markers of chromosome 2. Loci

Cross

9 X

W 100 x RLT 40

w 130

Recombination frequency

0'

X

RLT 40

an/Rap/R-

er/Rpy/Rgl11Rhy2/Rcer2/Rbp/Rtt31R-

0.4740 ± 0.0570 0.4477 ± 0.0552 0.1646 ± 0.0323 0.1608 ± 0.0319 0.3669 ± 0.0495 0.5516 ± 0.0620 0.4931 ± 0.0583 0.5048 ± 0.0591 0.2958 ± 0.0440

hy3/Rer/Rpy!R-

0.4032 ± 0.0466 0.3083 ± 0.0402 0.1883 ± 0.0310

the ak2 locus is located at about 36.0 eM from the er locus, 19.8 eM from py and 55.8 eM from the hyJlocus. Discussion

The mutant RLT 40, selected on LT-medium, was characterized by a 6-fold increase in free threonine in young plants,

255

and even by a slight accumulation of soluble lysine. Additional characterization of the mutant has revealed that RL T 40 had an altered AK-sensitivity towards the lysine feedback control, possibly due to a change in the lysine-binding site since Ki for lysine was 0.091 in the wild type and 0.456 in RLT 40. Therefore, the regulatory properties of AK were studied in detail both in the wild type and the mutant. In Arabidopsis, lysine is the major effector of AK-activity: the enzyme can be inhibited up to 68 %. Threonine exerted only an inhibition of 15 %, and 15 to 20% of the global activity remained insensitive to both amino acids. The regulatory properties of the Arabidopsis enzyme are comparable with those of AK from other plant species (Azevedo et al., 1992; Frankard et al., 1991; Kochhar et al., 1986; Rognes et al., 1983; Wilson et al., 1991). However, Arabidopsis differs from these species in having 15 to 20 % of its total enzyme activity insensitive to lysine and threonine. Separation of the isoforms was attempted by ion-exchange chromatography using a Mono Q column with a FPLC system. The obtained elution profile strongly suggested the presence of three isoforms in Arabidopsis: AK I threoninesensitive, AK II lysine-sensitive and AK ill insensitive to lysine and threonine. These results can be compared with those obtained for barley, where three independant AKisoenzymes with different regulatory properties were separated (Bright et al., 1982; Arruda et al., 1984). As in barley, the threonine-sensitive peak was the first one to be eluted. However, in Arabidopsis the threonine-form represented up to 50% of the global activity, whereas in barley only 10% was identified as threonine-sensitive. Furthermore, in barley no insensitive Ak-form has been found as in Arabidopsis. In maize, three AK-forms have also been recently found: one sensitive to lysine, one sensitive to lysine and SAM and one sensitive to threonine (Azevedo et al., 1992). The native Mr of the lysine-sensitive AK-form was approximately 238,000, while the threonine-form had a Mr of circa 183,000. The molecular weight of the lysine-form found in Arabidopsis is situated in the range from the ones obtained in carrot (Relton et al., 1988) and maize (Dotson et al., 1989), whereas the value of the threonine-form is comparable with the one found in maize (Azevedo et al., 1992). However, the occurring variation in molecular weight may be due to the Mr determination by different methods (gel filtration versus electrophoresis on native gels). AK of RLT 40 also separated into three different activity peaks by ion-exchange chromatography. AK I (threoninesensitive) and AK ill (insensitive) remained unchanged in their regulatory properties, whereas AK II was only 20% inhibited by 10 mM lysine, instead of 55% in the wild type. In contrast, the threonine overproducer RLT 70 of tobacco was characterized by a completely desensitized lysine-isoform (Frankard et al., 1991). In barley, three mutants resistant to lysine plus threonine were selected (Bright et al., 1982; Rognes et al., 1983). They were characterized by an AK with greatly decreased sensitivity to inhibition by lysine. Therefore, the phenotype of RLT 40, characterized by the resistance to LT-medium and enhancement of the soluble threonine pool, may be explained by assuming that under the LT-selective conditions, which are known to inhibit methionine synthesis, the decreased sensitivity of one AK-

256

BEm HEREMANs and MicHEL jACOBS

isoform to lysine enables the mutant plants to produce methionine still sufficient for growth. Since AK-lysine in the wild type represented 42% of the global enzyme activity and this isoform was inhibited by 55%, 22% of the total enzyme activity was affected by lysine. On the other hand, in RLT 40 the activity of AK-lysine was inhibited by 20%. As a consequence, not more then 8% of the AK-activity was sensitive to lysine since the relative proportion of the different isoforms did not significantly differ from the one observed in the wild type. The decreased sensitivity of AK II to lysine feedback was associated with a single nuclear dominant mutation, conferring resistance to LT. Linkage analysis revealed that the corresponding ak21ocus is located on chromosome 2 at 19.8 eM from py, 36.0 eM from the er locus and behaved as unlinked with hy3. In the near future the mutant will be crossed to two other mapping lines (NW128 and NW147) specific to chromosome 2 with markers situated in the vicinity of the ak2 locus, to precisely locate its position on the genetic map. In a following step, the localization of the ak2 locus with regard to closely linked RFLP markers will be developed. This may allow for the isolation of the gene ak2 coding for a lysine-sensitive AK-form. In plants until now, the only gene that has been cloned encodes the bifunctional aspartate kinase-homoserine dehydrogenase isozyme, which is regulated by threonine (Wilson et al., 1991; Ghislain et al., 1994). Due to the difficulties in purifying a lysine-sensitive AK to homogeneity, a strategy based on chromosome walking and physical mapping could represent the right approach for cloning the ak2 gene, which plays an essential role in the aspartate pathway. The small genome of Arabidopsis and the availability of high-density RFLP (Chang et al., 1988; Nam et al., 1989) and RAPD maps (Reiter et al., 1992) make such astrategy feasible. Recently, two cases of such an approach have been found to be successful in Arabidopsis thaliana (Arondel et al., 1992; Giraudat et al., 1992). Acknowledgements

We wish to thank I. Verbruggen and S. Vernaillen for technical assistance with amino acid analysis and Prof. Dr. L. Kanarek and E. Czerwiec for FPLC use and assistance. This investigation was supported by a grant provided to B. H. by the Belgian National Fund for Scientific Research and by «Geconcerteerde Akties» {92-97-13) from the Belgian governement.

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