Identification of a Monofunctional Aspartate Kinase Gene of Arabidopsis Thaliana with Spatially and Temporally Regulated Expression

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Genes Genet. Syst. (2001) 76, p. 189–198 Identification of a monofunctional aspartate kinase gene of Arabidopsis thaliana with spatially and temporally regulated expression

Yasushi Yoshioka*, Shunsuke Kurei, and Yasunori Machida Division of Biological Science, Graduate School of Science, Nagoya University Furo-cho, Chikusa-ku, Nagoya 464-8602, JAPAN

(Received 19 March 2001, accepted 24 April 2001)

We screened a gene trap library of Arabidopsis thaliana and isolated a line in which a gene encoding a homologue of monofunctional aspartate kinase was trapped by the reporter gene. Aspartate kinase (AK) is a key enzyme in the biosynthsis of aspartate family amino acids such as lysine, threonine, isoleucine, and methionine. In plants, two types of AK are known: one is AK which is sensitive to feedback inhibition by threonine and carries both AK and homoserine dehydrogenase (HSD) activities. The other one is monofunctional, sensitive to lysine and synergistically S-adenosylmethionine, and has only AK activity. We concluded that the trapped gene encoded a monofunctional aspartate kinase and designated as AK-lys3, because it lacked the HSD domain and had an amino acid sequence highly similar to those of the monofunctional aspartate kinases of A. thaliana. AK-lys3 was highly expressed in xylem of leaves and hypocotyls and stele of roots. Significant expression of this gene was also observed in trichomes after bolting. Slight expression of AK-lys3 was detected in vascular bundles and mesophyll cells of cauline leaves, inflorescence stems, sepals, petals, and stigmas. These results indicated that this aspartate kinase gene was not expressed uniformly but in a spatially specific manner.

phosphate by ATP (Fig. 1) (Bryan, 1990). In plants, two INTRODUCTION types of aspartate kinase have been identified so far The vascular bundle in higher plants is essential to their (Kochhar et al., 1986; Dotson et al., 1989; Azevedo et al., growth; its major functions are transport of water, nutri- 1992). One is sensitive to feedback inhibition by threo- ents, and signal molecules and support of plant body. nine (for review Galili, 1995). This type is bifunctional Despite the importance of the vascular tissue in plant with both aspartate kinase and homoserine dehydroge- growth, the molecular mechanisms of development of the nase (HSD) activities; the HSD activity resides at the vascular bundle and its physiological functions are still C-terminal of the AK sequence (Zhang et al., 1990; Ghis- incompletely understood. lain et al., 1994; Muehlbauer et al., 1994). The other type To investigate the genetic regulation of the formation of aspartate kinase is monofunctional and sensitive to and physiological functions of the vascular bundle, we feedback inhibition by lysine and synergistically S-adeno- screened a gene trap library of Arabidopsis thaliana to sylmethionine (Rognes et al., 1980). One copy of the identify genes expressing exclusively or primarily in vas- threonine-sensitive aspartate kinase gene (AK/HSD) cular tissue. (Ghislain et al., 1994) and two copies of the monofunctional We have screened 1,679 lines of the library we made and aspartate kinase genes have been cloned in Arabidopsis isolated a line in which a gene encoding a homologue of thaliana (AK-lys1 and AK-lys2, which was formerly desig- monofunctional aspartate kinase (AK) was trapped by the nated cArb-AK-lys) (Frankard et al., 1997; Tang et al., reporter gene. 1997). Aspartate kinase is a key enzyme in the biosynthsis of In the biosynthesis pathway of the aspartate family aspartate family amino acids such as lysine, threonine, amino acids, the enzymes dihydrodipicloinate synthase isoleucine, and methionine, catalyzing the first step of the (DHPDS) and HSD are also sensitive to feedback inhibi- synthesis through the activation of aspartate to aspartly- tion by the end products (Fig. 1). Analysis of various plant mutants possessing modified, feedback-insensitive en- Edited by Eiichi Ohtsubo zymes have shown that DHDPS plays a major regulatory * Corresponding author. E-mail: [email protected] role in lysine synthesis and that the AK rate limits the 190 Y. YOSHIOKA et al.

about the spatial and temporal expression pattern of monofunctional AK. In this report, we describe the expression pattern of the newly identified monofunctional AK gene designated AK-lys3 of A. thaliana. We found that AK-lys3 was highly expressed in xylem of leaves and hypocotyls and stele of roots. This gene was also highly expressed in trichomes after bolting. Weak expression of AK-lys3 was detected in veins and mesophyll cells of cauline leaves, inflorescence stems, sepals, petals, and stigmata.

MATERIALS AND METHODS Plasmid construction All plasmid manipulations were carried out according to standard procedures (Sambrook et al., 1989). The binary plasmid vector pNU74 used to generate the gene trap library was constructed as follows. A HindIII-EcoRI fragment of pARK22 (Anzai, unpublished results) containing cauliflower mosaic virus (CaMV) 35S promoter, the phosphinothricin acetyl transferase gene (bar) (Thompson et al., 1987), and the poly-adenylation site of the nopalin synthase gene (Tnos) was ligated into Fig. 1. Schematic representation of the biosynthetic pathway of pSHG396 (Takeshita et al., 1987) which had been digested aspartate family amino acids. Only the major key enzymes are with HindIII and EcoRI. The resulting plasmid was di- indicated. AK, aspartate kinase; HSD, homoserine dehydroge- gested with HindIII, treated with T4 DNA polymerase nase; DHDPS, dihydrodipicloinate synthase. (Takara Shuzo, Kyoto, Japan) to fill in the ends, and then digested with BamHI to eliminate the CaMV 35S pro- synthesis of threonine (Galili, 1994; Galili et al., 1995, and moter of bar. The linearized plasmid was ligated with an references therein). EcoRI-BamHI fragment of pKU3 (Velten et al., 1984) con- The biosynthetic pathway of aspartate family has been taining the 1'2' dual promoter (P1'2') whose EcoRI end had studied mostly at the biochemical level; several lines of been filled in by the treatment with T4 DNA poly- evidence have suggested that this pathway is also subject merase. A NarI-EcoRI fragment of this plasmid that con- to complex developmental and environmental regulation. tained P1'2'-bar and Tnos was ligated into pUC118 (Vieira For instance, the expression of the AK/HSD gene of A. and Messing, 1987), which had been digested with ScaI thaliana is subject to spatial and temporal transcriptional and EcoRI to give pUC118-P1' bar. control in vegetative tissue, flowers, and developing The plasmid pIG121-SA3 is a derivative of pIG121 seeds. Upon exposure of young etiolated seedlings to (Ohta et al., 1990) that has two artificial splicing acceptor light, expression of this gene was significantly up-regu- signals in addition to the original one at the 3' end of the lated in cotyledons and diminished in the hypocotyl intron of β-glucuronidase gene (GUS, see Fig. 2b). The (Zhu-Shimoni et al., 1997). The expression of DHDPS artificial splicing acceptor signals were synthesized as genes was also shown to be tissue-specific (Vauterin et al., oligonucleotides and inserted into the SalI site of the GUS 1999; Craciun et al., 2000; Sarrobert et al., 2000). Two gene. Plasmid DNA of pUC118-P1' bar was digested with copies of genes encoding DHDPS, DHDPS-1 and DHDPS- EcoRI to linearize it and then inserted into the EcoRI site 2, are present on the genome of A. thaliana (Vauterin of pIG121-SA3 to give pNU48. A 4.9-kb of PstI fragment et al., 1999; Craciun et al., 2000; Sarrobert et al., 2000). of pBI121 containing right border (RB) of T-DNA was One of these, DHDPS-1 was shown to be expressed in cloned into PstI site of pUC119. A SphI-HindIII frag- meristems, young vascular bundles of roots, vascular ment of this plasmid containing RB was replaced by the bundles of stem and leaves, carpels, style, stigma, synthetic RB sequence; the nucleotide sequences adjacent develping embryos, tapetum of young anthers, and pollens to RB had been altered to 5'-AAACACAGATCTAAGCTT- (Vauterin et al., 1999). Although there are some discrep- 3' to introduce BglII and HindIII sites. The resulting ancies in the reports about the expression of DHDPS-2, plasmid was digested with PstI and BglII and then ligated DHDPS-2 was also expressed in a tissue-specific manner with the BamHI-PstI fragment of pNU48 containing GUS, (Craciun et al., 2000; Sarrobert et al., 2000). Tnos, replication origin and ampicillin resistance gene of Despite the extensive knowledge about expression pat- pUC118, P1'2' -bar-Tnos, and LB to give pNU74. The T- terns of AK/HSD and DHDPS isozymes, little is known DNA region of pNU74 was depicted in Fig. 2a. An aspartate kinase gene of Arabidopsis thaliana 191

incubated overnight in a solution containing 1 mg/ml of 5- bromo-4-chloro-3-indoxyl-β-D-glucuronic acid, cyclohexy- lammonium salt in 100 mM potassium phosphate, pH7.0, 0.1% TritonX-100, and 10 mM EDTA at 37°C using a method described by Jefferson et al. (Jefferson, 1987). Cross sections 10 µm thick were made from hypocotyl samples that had been fixed in FAA (5% formaldehyde, 5% acetic acid, 63% ethanol), dehydrated in a graded ethanol series, and embedded in Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany) after the staining of GUS. These sections were stained with Safranin O and observed under a light microscope.

Rapid amplification of 5' cDNA ends reverse tran- scription-PCR (5'-RACE RT-PCR) Total RNA was prepared from rosette leaves of NUGeT539 using an RNeasy mini kit (QIAGEN, Hilden, Germany). Poly A+ RNA was purified from total RNA using Dynabead (Dynal A. S., Oslo, Norway) according to the procedure recom- mended by the supplier. A GUS fused mRNA was amplified by 5'-RACE RT-PCR by Marathon cDNA amplification kit (Clontech Laboratories, Palo Alto, CA, USA). We used oligonucleotides, GSP1 (5'-taaagacttcgcgctgataccagacg-3') and GSP2 (5'-ccaccaacgctgatcaattccacagt-3') complementary to GUS as nested primers. An amplified DNA frag- Fig. 2. Structure of the T-DNA region of the gene trap binary ment was cloned into the pGEM-T Easy plasmid (Promega plasmid, pNU74. (a) Schematic representation of the T-DNA of Co., Madison, WI, USA). pNU74. Right border, RB; left border, LB; β-glucuronidase gene with intron, GUS; poly-adenylation site of the nopalin synthase Plasmid rescue Genomic DNA was prepared from gene, Tnos; replication origin of pUC plasmid, pUCori; ampicillin NUGeT539 by an procedure described previously (Murray resistant gene of pUC118, Ampr; 1'2' dual promoter, P1'; basta resistance gene, BAR. The intron of GUS is shown by a shaded and Thompson, 1980). A half microgram of genomic DNA box. The DNA region used as a probe for Southern hybridization was digested by HindIII that does not cut the T-DNA analysis is indicated by a thick horizontal line. SacI recognition region of pNU74. This digested DNA was ligated by sites are also indicated. (b) Nucleotide sequence of splicing do- T4 DNA ligase and introduced into E. coli DH10B by nor and splicing acceptor signals of the GUS gene. Right border electroporation. (RB) is boxed. Splicing donor signal (SD) and splicing acceptor signal (SA) sequences are underlined. A cryptic splicing acceptor signal is indicated by double underlining. The intron of the Database search Sequence databases were searched GUS is shaded. A synthetic oligonucleotide used to generate two using the National Center for Biotechnology Information artificial splicing signals is indicated by italics. (NCBI) network BLAST service (Altschul et al., 1990; Madden et al., 1996). The tblastn search of the NCBI non-redundant database used the BLOSUM62 matrix. Transformation of Arabidopsis thaliana Arabi- dopsis thaliana ecotype Columbia was used for the con- Complementation of an aspartate kinase mutant of struction of the gene trap library and transformed in E. coli Complementary DNA of AK-lys3 mRNA includ- planta essentially according to the method described by ing entire coding sequence was amplified by PCR from Galbiati et al. (2000). Agrobacterium tumefaciens GV- cDNA that was prepared from 17-day-old plants using two 3101 containing pNU74 was used for the transformation. synthetic oligonucleotides, E10AK5SD (5'-gaggtgagt- Basta-resistant plants were selected on soil as follows. aatggcggcttcaatgcagttc-3') and E10AK3 (5'-ctagctagt- T1 seed was vernalized and planted directly in soil con- actttcagaccttgcagg-3'), as primers. E10AK5SD corre- taining 0.02% of Basta (AgrEvo, Frankfurt, Germany). sponds to position 88115 to 88089 of BAC clone F28J7 Three foliar sprays of 0.02% Basta were applied at 10, 11, (GenBank accession no. AC010797) and contains the and 13 days after germination. Shine-Dalgalno (SD) sequence at the 5' end (5'-gagg-3'). E10AK3 corresponds to position 85391 to 85422 of the Histochemical staining of GUS For histochemical same BAC clone. The amplified DNA fragment was analysis, whole tissues or sections (150 µm thickness) were cloned into pGEM-T Easy plasmid (Promega Co.) in the 192 Y. YOSHIOKA et al.

direction of the sense strand, so that it could be tran- detected in mesophyll cells (Fig. 3e). It was, however, scribed by the lacZ promoter to give pNU330. The direc- possible that the blue dye in mesophyll cells was caused tion of the cDNA was confirmed by nucleotide sequencing. by diffusion from the veins since the GUS expression in AKs of E. coli are encoded by three genes designated thrA, veins were extremely high in old leaves (Fig. 3e). After metL, and lysC. A triple mutant of these three genes, bolting, significant activity of GUS became visible in the Gif106M1 (thrA1101, ilvA296, metLM1000, lysC1001, arg- trichomes of leaves and inflorescence stems (Fig. 3g and 1000) (Thèze et al., 1974) was transformed by pNU330 and 3h). GUS was also expressed in veins and mesophyll cells streaked on M9 minimal agar plates containing 0.2% of cauline leaves (Fig. 3f), and there was slight expression lactose, 0.3 mM isoleucine, 0.3 mM valine, 0.3 mM me- throughout the entire region of the inflorescence stem thionine, 0.3 mM arginine, 2 mM of threonine, and 1mg/ (Fig. 3g). In flowers, the expression of GUS was detected ml thinamine · HCl. Lysine was added to 0.3 mM to the throughout the entire sepal, petals, stamens, and stigmata lysine containing M9 agar plates. Gif106M1 containing (Fig. 3i and 3j). a pGEM-T Easy vector plasmid that had been digested To investigate the region of the expression of GUS at with EcoRI and self-ligated was used as a negative con- the cellular level, we made sections of rosette leaves and trol. A thrA metL double mutant, Gif99 (thrA1101, hypocotyls. As shown in Fig. 3k and 3l, strong expression ilvA296, metLM1000, arg-1000) (Thèze et al., 1974) was of GUS activity was observed in xylem of rosette leaves used as a positive control. and hypocotyls.

Cloning of the gene that was trapped by GUS To RESULTS determine the copy number of the T-DNA that was inte- Isolation of a line with a strong GUS expression in grated into the genomic DNA of NUGeT539, Southern blot leaf veins To identify genes that were expressed in vas- analysis was performed. Genomic DNA of NUGeT539 cular tissues, we made a gene trap library of Arabidopsis was digested with SacI, fractionated on agarose gel, and thaliana Col-0 using the binary plasmid pNU74. This plas- blotted on a nylon membrane. This membrane was hy- mid contains a GUS without a promoter and the transla- bridized with the GUS-Tnos DNA fragment indicated by tional initiation codon as a reporter in the T-DNA region the solid line in Fig. 2a. In this analysis, three bands (Fig. 2). We examined 1,679 T2 plants for the expression were expected per one copy of the T-DNA, because two of GUS by histochemical staining two weeks after sowing, SacI recognition sites are present in the T-DNA region as described in MATERIALS AND METHODS, and found (Fig. 2a). One of these bands will be 4.6 kb in length and a line designated NUGeT539 in which the significant GUS represent the internal SacI fragment of the T-DNA. As activity was observed in the veins of rosette leaves (Fig. shown in Fig. 4a, three bands of 8 kb, 4.6 kb, and 1.2 kb in 3e). No abnormality in growth, shape, morphology of vas- length were detected in the Southern blot analysis of cular bundles in veins, or fertility was observed in the NUGeT539, indicating that one copy of the T-DNA was plants homozygous for insertion of GUS into this line integrated into the genomic DNA of NUGeT539. when compared with wild-type plants (data not shown). Complementary DNA corresponding the gene that was trapped by the GUS reporter gene was amplified by 5'- Expression patterns of GUS in NUGeT539 The spa- RACE RT-PCR, as described in MATERIALS AND tial and temporal expression patterns of GUS in NUGe- METHODS. We cloned this chimeric cDNA in the T539 were examined at various stages of development by pGEM-T Easy vector plasmid and determined its nucle- histochemical staining using plants homozygous for the otide sequence. Comparison of the predicted amino acid insertion of GUS. As shown in Figure 3a, GUS activity sequence of the chimeric cDNA with the GenBank data- became detectable around the stele of the upper part of base showed that the reading frame of the GUS gene was root in 4-day-old seedlings. This expression pattern in fused with that of a putative aspartate kinase gene on roots was preserved throughout plant development: GUS chromosome III of A. thaliana (GenBank accession no. activity was detected in the upper parts of the primary AAF03452, Fig. 4b). We designated this gene as AK-lys3, and lateral roots but not in the lower parts (Fig. 3c, d). because its predicted amino acid sequence shared high When the first and second leaves had expanded, GUS identity with those of monofunctional AKs of A. thaliana activity was detected in the vascular bundles of the hypo- (see below). To determine the T-DNA integration site in cotyl and petioles and in major veins of cotyledons (Fig. AK-lys3, the T-DNA and the adjacent genomic DNAs was 3b). After this developmental stage, GUS activity began rescued from the NUGeT539 as described in MATERIALS to be detected in major veins of rosette leaves. In young AND METHODS. The analysis of the nucleotide se- rosette leaves, GUS activities were detected only in the quences of the junctions between the ends of the T-DNA major veins (Fig. 3e). Then they were detected with and the genomic DNA revealed that the T-DNA was inte- aging in both major and minor veins but not in veinlets grated into the fifth intron of AK-lys3 (Fig. 4b). (Fig. 3e). In old leaves, a slight expression of GUS was Comparison of the nucleotide sequences of the chimeric An aspartate kinase gene of Arabidopsis thaliana 193

Fig. 3. GUS activity in the NUGeT539. (a) Seedling at 4 days after germination. (b) Plantlet with expanding first and secocnd leaves. (c) Roots: vascular bundle of upper part of primary root shows high GUS expression, whereas lower part of primary root and lateral roots show no GUS expression. (d) Root tip of a primary root. (e) Leaves and roots of 23-day-old plants. (f) Cauline leaf. (g) Inflorescence stem. (h) Trichomes showing high GUS expression on resette leaf. (i) GUS expression in petals (pe) and sepals (sp) (j) GUS expression in stamens and stigma. Petals and sepals are removed. (k) Cross section (150 µm thickness) of mid vein of rosette leaf. (l) Cross section of hypocotyl (10 µm thickness). xy, xylem; ph, phloem. Bars represent 1 mm (a–d, f–j), 1 cm (e), and 0.01 mm (k, l). cDNA, the predicted AK-lys3 cDNA, and the GUS cDNA AK-lys2, which was formerly designated cArb-AK-lys) indicated that AK-lys3 was trapped by a cryptic splicing have been cloned in A. thaliana (Frankard et al., 1997; acceptor sequence in the intron of GUS (double underline Tang et al., 1997). Comparison of their amino acid se- in Fig.4c). quences with the predicted AK-lys3 one revealed that the highest identity score was between the amino acid Comparison of the predicted amino acid sequence sequences of AK-lys3 and AK-lys2 (80%). The identity of of the trapped gene with those of aspartate the amino acids between the sequences of AK-lys3 and AK- kinases One copy of a threonine-sensitive aspartate lys1 was also high (67%), but that between AK/HSD kinase (AK/HSD) gene (Ghislain et al., 1994) and two cop- (Ghislain et al., 1994) and AK-lys3 was only 26%. Fur- ies of lysine-sensitive aspartate kinase genes (AK-lys1 and thermore, AK-lys3 did not contain the amino acid se- 194 Y. YOSHIOKA et al.

quence that is similar to HSD. These results indicate that the AK-lys3 gene that was trapped in NUGeT539 encoded a monofunctional aspartate kinase. As shown in Fig. 5, the predicted amino acid sequence of AK-lys3 was 559 residues in length and contained three amino acid motifs that are highly conserved among aspartate kianses: the KFGG motif (positions 93 to 96 in Fig. 5) (Frankard et al., 1997; Gebhardt et al., 1999; Ghislain et al., 1994; Muehlbauer et al., 1994; Tang et al., 1997; Weisemann and Matthews, 1993), the DPR motif (position 323 to 325 in Fig. 5) (Ghislain et al., 1994), and the monofunctinal aspartate kinase box (TTLGRGGSD, amino acid position 289 to 295 in Fig. 5) (Frankard et al., 1997). Although the highest identity score of deduced amino acid sequence of AK-lys3 was with those of AK-lys1 and AK- lys2, its N-terminal region showed relatively low identity with the N-terminal of the other two AKs, suggesting that its N-terminal contained a transit peptide, as do the all other previously cloned plant AK sequences did (Weisemann and Matthews, 1993; Ghislain et al., 1994; Muehlbauer et al., 1994; Frankard et al., 1997; Tang et al., 1997; Gebhardt et al., 1999).

Complementation of an asparatate kinase mutant of E. coli by AK-lys3 To examine if AK-lys3 actually has aspartate kinase activity, we introduced the cDNA of AK- lys3 into a triple mutant of aspartate kinase genes (lysC, metL, thrA) of E. coli, Gif106M1, because the wild type of E.coli has three aspartate kinase genes: one for monofunctional aspartate kianse (lysC) and two for bifunctional aspartate kinases (metL and thrA) (Thèze et al., 1974). The cDNA of AK-lys3 was amplified and cloned into the pGEM-T Easy plasmid vector (Promega Co.) in the direction of the sense strand, so that it could be transcribed by the lacZ promoter as described in MATERIALS AND METHODS. The resulting plasmid was designated pNU330 and introduced into Gif106M1. As shown in Fig. 6, Gif106M1 cells harboring pNU330 were able to grow on M9 minimal agar plates without lysine containing 2 mM Fig. 4. Analysis of the T-DNA integration site in NUGeT539. threonine, 0.3 mM methionine, and 0.2% lactose, although (a) Southern blot analysis of NUGeT539. Total DNA of Gif106M1 harboring the vector plasmid alone was not NUGeT539 (lane 1) and wild type plants (lane 2) were digested with SacI and fractionated by 0.7% of agarose-TAE gel. Arrows able to grow on the same M9 agar plates. Although the indicate DNA fragments hybridized with the probe DNA shown growth of Gif106M1 was not fully restored by AK-lys3 in Fig. 2a. (b) Schematic representation of the genomic struc- (compare pNU330 with the positive control strain, Gif99), ture of AK-lys3. Deduced exons are represented by open boxes. our results indicate that AK-lys3 functioned as aspartate T-DNA was integrated between the 5th and the 6th exon in the kinase in E. coli cells. direction indicated. The length of T-DNA is not proportional to the scale bar in (b). (c) Nucleotide sequence of the chimeric cDNA of AK-lys3 and GUS (AK::GUS). Deduced nucleotide sequences Complexity of aspartate kinase genes in Arabi- and amino acid sequences of AK-lys3 (AK) and GUS (GUS) dopsis thaliana To estimate the complexity of the as- are shown. Identical nucleotides are indicated by vertical lines. partate kinase genes in Arabidopsis thaliana, we searched Junction of exon 5 and 6 of AK-lys3 is indicated above the se- the genomic DNA of A. thaliana by BLAST using the quence. Junction of intron-exon of GUS gene that was reported by Ohta et al.(1990) is indicated below the sequence. Double amino acid sequences of AK-lys1, AK-lys2, and AK-lys3 as underline and underlines indicate cryptic splicing acceptor signal queries (Altschul et al., 1990; Madden et al., 1996). We and splicing acceptor signals, respectively. found two proteins with significant similarities to AK-lys1, AK-lys2, and AK-lys3. One was AK/HSD, that was re- An aspartate kinase gene of Arabidopsis thaliana 195

Fig. 5. Alignment of amino acid sequences of AK-lys3, AK-lys2, and AK-lys1. Identical residues are shaded. Putative transit pep- tides are indicated by wavy lines. KFGG motif, monofunctional AK box, and DPR motif are indicated by horizontal lines. The T-DNA integration site is indicated by an arrowhead. Alignment was performed using CLUSTAL W program (Thompson et al., 1994). ported by Ghislain et al. (1994), and the other was an AK/ shown) indicating that the AK/HSD-like protein was the HSD-like protein that has not been published (GenBank second AK/HSD gene of A. thaliana. accession no. CAB78973). The identity of amino acids be- We also examined the complexity of the AK/HSD tween AK/HSD and AK/HSD-like was high (76%, data not gene in the entire genome of A. thaliana by BLAST search 196 Y. YOSHIOKA et al.

because of the loss of the monofunctional AK box and the DPR motif, which are thought to be important for AK activity. However, no differences were observed in growth, development, or fertility. This can be explained by the redundancy of the AK genes. AK-lys1 and AK-lys2 may be expressed in the same regions as AK-lys3. Al- ternatively, the aspartate family amino acids may be transferred from the regions where AK-lys1 or AK-lys2 are expressed. Some of the key enzymes for the synthesis of these amino acids have been reported to be expressed in a tissue-specific manner (Zhu-Shimoni et al., 1997; Vauterin et al., 1999; Craciun et al., 2000; Sarrobert et al., 2000). The expression pattern of GUS in NUGeT539 also indicated that AK-lys3 was not expressed uniformly but spe- cifically in xylem of rosette leaves and hypocotyls and stele of roots before bolting. This gene was also expressed in the entire regions of the cauline leaves, inflorescence stems, sepals, petals, stigmas, and trichomes after bolting. To our knowledge, this is the first histological Fig. 6. Complementation of an AK mutant of E. coli by AK- analysis of the expression of the Lys-sensitive aspartate lys3. A triple mutant of AK, Gif106M1 (thrA, metL, lysC) (Thèze kinase gene in plants. We believe that the expression et al., 1974), harboring pNU330, which contains the cDNA of AK- pattern of GUS observed in NUGeT539 reflected that of lys3, was streaked on M9 minimal agar plates with (lower right AK-lys3, because the chromosomal position and the 5' panel) or without (lower left panel) lysine. Gif106M1 cells harboring the vector plasmid alone or no plasmid were streaked on untranslated region of the GUS fused AK-lys3 gene were the same plates as a negative control. Gif99 is a double mutant unchanged. This chimeric gene is probably under the of AK (thrA, metL) used as a positive control (Thèze et al., same transcriptional control as the native AK-lys3.We 1974). Isoleucine (0.3 mM), valine (0.3 mM), methionine cannot, however, rule out the possibility that the expres- (0.3.mM), and threonine (2 mM) were supplied to the plates be- sion pattern of GUS does not exactly represent that of cause of the HSD deficiency of Gif106M1 and Gif99. Lactose (0.2%) was added to the plates as a carbon source to induce the AK-lys3. It is possible that the 3' downstream region of expression of AK-lys3 cDNA from the lac promoter. AK-lys3 that was separated from the 5' upstream region by the insertion of the T-DNA may contain the cis element crucial for the temporal and/or spatial expression pattern using the amino acid sequences of AK/HSD and AK/HSD- of the AK-lys3. Additionally, the stability of the fusion like as queries. No amino acid sequences significantly transcript between AK-lys3 and GUS may differ from that similar to either AK/HSD or AK/HSD-like was identified of the native gene. other than the three monofunctional AKs. These results One of the unexpected features of the expression of indicate that three monofunctional AK genes and two AK/ AK-lys3 was its slight expression in mesophyll cells in HSD genes are present on the genome of A. thaliana. rosette leaves. Because proteins participating in photo- synthesis are synthesized in mesophyll cells of expanding leaves, the synthesis of amino acids should be active in DISCUSSION mesophyll cells. Our results, however, indicated that the We have shown that a gene with significant expression AK-lys3 gene, which encodes the key enzymes in the in xylem of leaves and hypocotyls and stele of roots encodes biosynthesis of the aspartate family amino acids, was a protein highly homologous to the monofunctional aspar- expressed preferentially in xylem of veins and only slightly tate kinase of A. thaliana. This gene, designated AK- in mesophyll cells in rosette leaves. Interestingly, the lys3, is probably a Lys-sensitive aspartate kinase like the DHDPS-1 gene has been shown to be expressed in veins of other monofunctional aspartate kinases, because it lacks leaves but not in mesophyll cells (Vauterin et al., the HSD domain characteristic of threonine-sensitive 1999). The expression of the DHDPS-2 gene, the other aspartate kinases. Two genes for monofunctional AKs gene encoding DHDPS, does not seem to be highly ex- have been previously reported. AK-lys3 is the third gene pressed in mesophyll cells either, although there are some encoding a monofunctional AK. discrepancies in the reports about the expression of The T-DNA of pNU74 was inserted into the fifth intron DHDPS-2 (Craciun et al., 2000; Sarrobert et al., of the AK-lys3 gene (Fig. 4b and Fig. 5). In consequence, 2000). The slight expressions of DHDPS genes in meso- we can expect that AK-lys3 loses its function in NUGeT539 phyll cells indicate that the biosynthesis of lysine would An aspartate kinase gene of Arabidopsis thaliana 197 be low in leaf mesophyll cells and are consistent with our and Jacobs, M. (1994) Molecular analysis of the aspartate results. The physiological reasons for such low levels of kinase-homoserine dehydrogenase gene from Arabidopsis thaliana. Plant Mol. Biol. 24, 835–851. lysine biosynthesis in mesophyll cells remain to be exam- Jefferson, R. (1987) Assaying chimeric genes in plants: The Gus- ined. gene fusion system. Plant Mol. Biol. Rep. 5, 387–405. There are some regions where the expression of AK-lys3 Kochhar, S., Kochhar, V. and Sane, P. (1986) Isolation, charac- was detected but those of DHDPS-1 and DHDPS-2 were terization and regulation of isoenzymes of aspartate kinase not, such as roots other than the root tip, hypocotyls, se- differentially sensitive to calmodulin from spinach leaves. Biochim. Biophys. Acta 880, 220–225. pals, and petals (Vauterin et al., 1999; Craciun et al., 2000; Madden, T. L., Tatusov, R. L. and Zhang, J. (1996) Applications Sarrobert et al., 2000). These results seem to be incon- of network BLAST server. Methods Enzymol. 266, 131–141. sistent with the fact that both of these enzymes are critical Muehlbauer, G. J., Somers, D. A., Matthews, B. F. and Gengen- for lysine biosynthesis. No other gene encoding a protein bach, B. G. (1994) Molecular genetics of the maize (Zea mays significantly similar to DHDPS has been found in the ge- L.) aspartate kinase-homoserine dehydrogenase gene family. Plant Physiol. 106, 1303–1312. nome of A. thaliana by BLAST search. Consequently, the Murray, M. G. and Thompson, W. F. (1980) Rapid isolation of high substrate for DHDPS, L-aspartate-β-semialdehyde, might molecular weight plant DNA. Nucleic Acids Res. 8, 4321– be channeled into the threonine-isoleucine-methionine 4325. synthesis pathway in such regions. Alternatively, L-as- Ohta, S., Mita, S., Hattori, T. and Nakamura, K. (1990) Construc- β partate-β-semialdehyde or aspartly-phosphate (the me- tion and expression in tobacco of a -glucuronidase (GUS) reporter gene containing an intron within the coding se- tabolite of AK) might be transferred to a region where the quence. Plant Cell Physiol. 31, 805–813. DHDPS genes are expressed. Rognes, S. E., Lea, P. J. and Miflin, B. J. 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