JXB Advance Access originally published online on June 1, 2007
Journal of Experimental Botany 2007 58(10):2653-2660; doi:10.1093/jxb/erm100
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Characterization of monofunctional aspartate kinase genes in maize and their relationship with free amino acid content in the endosperm
1Department of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA
2Department of Plant Science, Weizmann Institute, 76100 Rehovot, Israel
To whom correspondence should be addressed. E-mail: Larkins{at}Ag.Arizona.edu
Received 2 March 2007; Revised 6 April 2007 Accepted 17 April 2007
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Abstract |
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A quantitative trait locus has previously been identified in maize (Zea mays L.) that influences the level of free amino acids in the endosperm, especially those from the aspartate pathway: lysine, threonine, methionine, leucine, and isoleucine. Because this locus occurs in a region of the genome containing ask2, a monofunctional aspartate kinase, the nature of the monofunctional aspartate kinase genes in the parental inbreds, Oh545o2 and Oh51Ao2, was investigated. Two genes, Ask1 and Ask2 were isolated, and Ask2 was mapped to the ask2 locus. Nucleotide sequence analysis of the Ask2 alleles from Oh545o2 and Oh51Ao2 showed they differ by one amino acid. Both alleles complemented a yeast aspartate kinase mutant, hom3, and based on the growth of the yeast mutant it appeared that Ask2-Oh545o2 produces an enzyme with greater total activity than that encoded by the Oh51Ao2 allele. The results suggest that the higher level of free amino acids derived from the aspartate pathway in Oh545o2 endosperm results from a single amino acid change in the ASK2 enzyme that has pleiotropic effects on its activity.
Key words: Aspartate kinase, endosperm, lysine, opaque2, Zea mays
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The aspartate pathway in higher plants plays an essential role in the regulation of the biosynthesis of several essential amino acids, including lysine, which is most limiting in the endosperm of maize (Zea mays L.) and other cereals. The first key step of this pathway is regulated by aspartate kinase (AK, EC 2.7.2.4 [EC] ), which catalyses the conversion of aspartate to β-aspartyl phosphate and is feedback-regulated by several end-products, including lysine and threonine (Bryan, 1990; Galili, 1995). Based on their biochemical properties, there are at least two isoforms of AK in plants, including lysine-sensitive and threonine-sensitive enzymes (Dotson et al., 1989; Azevedo et al., 1992a, b; Muehlbauer et al., 1994a, b). The lysine-sensitive isoform is primarily a monofunctional AK, while the threonine-sensitive isoforms are bifunctional aspartate kinase-homoserine dehydrogenase (AK-HSDH) enzymes. In maize, two mutant loci encoding monofunctional AK, ask1 and ask2, were identified by genetic screening (Diedrick et al., 1990; Dotson et al., 1990b; Muehlbauer et al., 1994a), and the enzymes they encode are feedback-inhibited by lysine (Dotson et al., 1989; Azevedo et al., 1992a). Mutations at these loci result in AKs that are less sensitive to lysine and result in the over-production of free lysine, threonine, methionine, and isoleucine (Dotson et al., 1990b; Muehlbauer et al., 1994a). Three bifunctional AK-HSDH cDNA clones have been isolated; two were mapped to the long arm of chromosome 2 and the short arm of chromosome 4 (Muelhbauer et al., 1994b). These genes appear to encode threonine-sensitive isoforms of AK (Azevedo et al., 1992b; Muehlbauer et al., 1994b). Three monofunctional AK genes were cloned and characterized in Arabidopsis thaliana (Frankard et al., 1997; Tang et al., 1997; Yoshioka et al., 2001), and while the biochemical features of maize monofunctional AK genes have recently been described (Anzala et al., 2006) the nucleotide sequences of these genes were not reported.
In a study on the variation of free lysine content in Oh545 opaque2 (o2) and Oh51Ao2 endosperm, a QTL was identified on the long arm of chromosome 2 with a map position that is coincident with the ask2 locus (Wang and Larkins, 2001). It was documented that the Oh545 inbred contains a higher level of free amino acids than Oh51A, and this difference is enhanced by the o2 mutation (Moro et al., 1996; Wang and Larkins, 2001). Biochemical studies suggested that allelic variation at the ask2 locus could account for the difference in free lysine content between Oh545o2 and Oh51Ao2 (Wang et al., 2001b). However, there was insufficient evidence to validate Ask2 as the candidate gene responsible for the QTL. The cloning and sequence analysis of two maize genes encoding monofunctional AK, Ask1 and Ask2 is reported here, and it is shown that the Ask2 gene is tightly linked to the QTL on chromosome 2 that is associated with a high endosperm lysine content. There is one amino acid difference between the Ask2 alleles in Oh545o2 and Oh51Ao2, but it is unclear how this affects AK activity or leads to the higher level of AK pathway free amino acids in endosperm of the Oh545o2 inbred.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Plant materials and mRNA preparation
cDNA clones encoding the Ask1 and Ask2 sequences in B73, and Ask2 from Oh545 and Oh51A, were obtained by 5'-rapid amplification of cDNA ends (5'-RACE) and RT-PCR using RNA from leaf and endosperm tissue. RNA was extracted from young shoot tissue of the B73 inbred by the trizol method, according to the manufacturer's instructions (Life Technology, Grand Island, NY); Poly(A) RNA was extracted from 15 d after pollination (DAP) endosperms as described by Wang et al. (2001a) using a Poly(AT)-tract kit (Promega, Madison, WI).
Cloning of monofunctional AK genes
The amino acid sequence of an Arabidopsis monofunctional AK gene (accession no. U62020
[GenBank]
; Tang et al., 1997) was used to BLAST search the maize EST database (http://www.zmdb.iastate.edu) and six ESTs (AW28241, AW066359
[GenBank]
, AI600499
[GenBank]
, AI629668
[GenBank]
, AI782896
[GenBank]
, and AI901511
[GenBank]
) with significant sequence identity to the Arabidopsis gene were identified. Most of these clones were obtained, and their nucleotide sequences were determined. Based on sequence alignment, the genes fell into three distinct groups (AI600499
[GenBank]
, AI629668
[GenBank]
, AI782896
[GenBank]
, and AI901511
[GenBank]
belong to one group, and AW282411
[GenBank]
and AW066359
[GenBank]
are each distinct). In collaboration with Pioneer Hi-Bred, five other clones were identified corresponding to putative monofunctional Ask genes in their EST database. These genes were found to belong to these same three groups. As none of these clones contained a full-length cDNA, 5'-RACE (Life Technology, Grand Island, NY) and RT-PCR techniques were used to obtain longer transcripts of each gene.
Ask1 was cloned from the AW066359 [GenBank] EST, which is the longest sequence among the clones in the Iowa State database (ZmDB); it is missing only part of the predicted transit peptide region at the 5' end. 5'-RACE was conducted with poly(A) RNA from the shoot meristem of the B73 inbred to obtain the complete coding sequence of the gene. The primers for 5'-RACE were: AW066359 [GenBank] -GSP1, 5'-GCATCCGCTCGGCTGACGCGA-3'; AW066359 [GenBank] -GSP2, 5'-CGAACTTCATGACGACGGTGAA-3'; and AW066359 [GenBank] -GSP3: 5'-CCCCGCCTGGGCATTCCTTG-3'. Since the AW066359 [GenBank] clone is from a cDNA library of the Illinois High Oil inbred, RT-PCR was used to clone the full-length cDNA from B73. PCR was conducted with a forward primer: 5'-GGGCCTGGCGGTGCGCTGCC-3' and a reverse primer: 5'-CACCGAAGCTCACCGCATTC-3'. For the 3' sequences, RT-PCR was performed using a primer: 5'-ATTTGGGCATCTCTGTTGAC-3’ and a 20-mer polyT-V primer. The PCR conditions were as follows: 94 °C for 2 min, then 30 cycles at 94 °C for 1 min, 54 °C for 40 s, and 68 °C for 1 min and 30 s; finally, 68 °C for a 5 min extension. The reaction solution contained the following: 5 µl of x10 PCR buffer (supplied with enzyme), 2 µl of 50 mM MgSO4, 1 µl of 10 mM dNTPs, 1 µl of 10 µM forward and reverse primers, 0.4 µl of Platinum Taq DNA Polymerase High Fidelity (Life Technology, Grand Island, NY), 1 µl of first strand cDNA, and double distilled H2O (DDH2O) to a final volume of 50 µl.
Ask2 was cloned similarly, based on the AW282411 [GenBank] EST by 5'-RACE and RT-PCR with a degenerate primer. The first strand cDNA was synthesized with B73 shoot meristem poly(A) RNA using an AK2-specific reverse primer, 5'-CAAGATGCAATTCCTTTACAAAG-3'. The reaction conditions were as follows: 5 µl of x10 PCR buffer, 2 µl of MgSO4, 1 µl of 10 mM dNTPs, 5 µl of 10 µM forward degenerate primer, 5'-ATGAARTTYGGSGGSTCXTC-3', 0.5 µl of 10 µM reverse primer, 5'-AGAGCTCATCAAGCTCCGAGACG-3', 2 µl of cDNA, 0.2 µl of Platinum Taq DNA Polymerase High Fidelity, and DDH2O to a final volume of 50 µl. The PCR conditions were as follows: 94 °C for 2 min, then 30 cycles at 94 °C for 1 min, 54 °C for 40 s, and 68 °C for 40 s, followed by 68 °C for 5 min. The PCR product was separated by electrophoresis in 1% agarose gel, and a single band was isolated and cloned with a TOPO-TA cloning system (Invitrogen, Carlsbad, CA92008). The 3'-UTR was cloned by RT-PCR using the forward primer, 5'-GCCATTAGCCGAGGATTTGA-3' and the polyT-V primer. The 5' sequence was extended by 5'-RACE with primers, 5'-CGCATCCTTCTTCTCGGCAACA-3' and 5'-GGCACCGACGGCTCGCTGACA-3'.
A third putative Ask contig was identified by four clones (AI600499 [GenBank] , AI629668 [GenBank] , AI782896 [GenBank] , and AI901511 [GenBank] ). However, each of them had a disrupted open reading frame, so this gene was not characterized further.
DNA extraction and Ask2 gene mapping
The Ask2 gene was mapped using 76 recombinant inbred lines (F6) derived by single seed decent from the F2 progeny of a cross between Oh545o2 by Oh51Ao2. Allele-specific primers from the 3'-untranslated regions (UTRs) of Oh545o2 and Oh51Ao2 were used to conduct PCR-based genotyping of the Ask2 gene. The reverse primers for the two alleles are: Ask2-Oh545o2, 5'-TCTCTAAACAAAATCCTAAGTAGTA-3', and Ask2-Oh51Ao2, 5'-CTCTAACCAAAATCCTAAGTAGTG-3'; the forward primer for both is: 5'-GCCATTAGCCGAGGATTTGA-3'. The genotypes of simple sequence repeat (SSR) markers (bnlg1138, bnlg1129, and bnlg1633) that flank the QTL region on the long arm of chromosome 2 were determined for this population as described by Wang et al. (2001a). The genetic distance and linkage map between the Ask2 gene and these SSRs were determined with the PC version of Map Manager QTXb15 (http://mapmgr.roswellpark.org/mmQT.html). The Morgan map function was used in this analysis.
DNA from young leaves of the parental and F6 lines was prepared by the CTAB method (Wang et al., 2001a) and diluted to a final concentration of 10 ng µl–1 for PCR reactions. For the Ask2-Oh51Ao2 allele, PCR reactions were initiated by denaturing the DNA at 94 °C for 2 min, followed by 35 cycles of PCR: 94 °C for 1 min, 57 °C for 40 s, and 72 °C for 30 s. The final cycle was extended at 72 °C for 5 min. Reactions were conducted in 0.2 ml thin-walled PCR tubes in a PTC-200 Peltier thermal cycler (MJ Research Inc., Watertown, MA). Each reaction contained 20 ng of maize DNA, 2.5 µl of x10 PCR reaction buffer, 0.5 µl of 50 mM MgCl2, 0.2 µl of 10 mM dNTPs, 0.2 µl of 10 µM of forward and reverse primers, and 0.2 units of Platinum Taq DNA polymerase; a final volume of 25 µl was made with sterile double-distilled H2O. For the Ask2-Oh545o2 allele, PCR reactions were initiated by denaturing the DNA at 94 °C for 2 min , followed by 35 cycles of PCR: 94 °C for 1 min, 56 °C for 40 s, and 72 °C for 30 s. The final cycle was extended at 72 °C for 5 min. Each reaction contained 20 ng of maize DNA, 2.5 µl of x10 PCR reaction buffer, 0.9 µl of 50 mM MgCl2, 0.2 µl of 10 mM dNTPs, 0.2 µl of 10 µM forward and reverse primers, and one unit of Platinum Hi-Fi Taq DNA polymerase; a final volume of 25 µl was made with double-distilled H2O. Following DNA amplification, the PCR products were separated by electrophoresis with 1% agarose in TAE buffer and visualized by staining with 0.01 µg of ethidium bromide per ml of gel.
Cloning of Ask2 alleles
The coding sequences of the Ask2 alleles of Oh545o2 and Oh51Ao2 were obtained by RT-PCR. For both genotypes, the same forward primer and reverse primers were used. The forward primer was 5'-TCCCCCGGGGGACTCACCGTCGTGATGAAGTTCGGCGGGT-3', and the reverse primer was 5'-CCCAAGCTTGGGCAGATGAATATCAAATCCTCGGCTA-3', which matches the region right after the stop codon.
Southern blot analysis
Twelve micrograms of genomic DNA from Oh545o2 and Oh51Ao2 was cut with restriction enzymes, separated by 0.8% agarose gel electrophoresis and blotted onto a nylon membrane. The PCR products corresponding to the 3'-UTRs of Ask1 and Ask2 from B73 were labelled with 
32P-dCTP by random priming and used as probes to hybridize with DNA on nylon membranes. Radioactivity hybridizing to the nylon membrane was detected by X-ray film exposure.
Functional complementation of maize Ask2 genes in yeast and AK assays
The Saccharomyces cerevisiae strain used in this study was 
a3hu (Mat ura3-52 hom3
GAL), which is derived from strain 1278b and carries a deletion in the HOM3 gene (Jones and Fink, 1982). This strain was constructed by Isabel Velasco and kindly donated by Pablo Marina Losada (Department of Genetics, Sevilla University, Spain). Transformation of the yeast ura3/hom3 mutant was performed using the shuttle vector p426 GPD, which contains the URA3 gene for selection and the GPD promoter to drive expression of the maize Ask2 genes (Mumberg et al., 1995). The Ask2-Oh545o2 and Ask2-Oh51Ao2 alleles were cloned into the pCRT7/NT TOPO expression vector (Invitrogen, Carlsbad, CA). The XbaI and HindIII fragment, including the Ask2 coding region and part of the vector sequence that contains 6XHisG and an Xpress epitope fused to the NH-terminus, was subcloned into the yeast expression vector, p426 GPD. The pMR3 vector, comprising the whole HOM3 gene of S. cerevisiae plus its promoter region (Martín-Rendón et al., 1993), was used as a positive control for strain complementation on medium lacking threonine. Yeast was transformed by lithium acetate treatment. Growth media for S. cerevisiae (SD, minimal medium; and YPD, complete medium) are described in Sherman et al. (1986). When necessary, minimal media was supplemented with the appropriate metabolites. Yeast cells were grown with vigorous stirring at 30 °C in minimal SD medium supplemented with appropriate amino acids. When the culture reached an OD600 of 0.7 (late exponential phase), the cells were harvested by centrifugation at 4 °C, washed twice, and resuspended in extraction buffer [500 mM phosphate buffer, pH 7.4, 50 mM KCl, 5 mM MgSO4, 10% (v/v) glycerol, 20 mM β-mercaptoethanol, 0.5 mM PMSF, 1:2000 protease inhibitor cocktail (Sigma), 1% (v/v) Tween 20 and 1 M urea] and disrupted by sonication followed by shaking the sample 1 h at 4 °C. Cell debris was removed by centrifugation at 10 000 g at 4 °C (Eppendorf, Germany). The supernatant was dialysed extensively against dialysis buffer (identical to extraction buffer except that it contained 0.2% (v/v) Tween 20 without urea) and the fresh crude protein extract was used for enzyme activity measurements.
Three and four independent yeast transformants expressing maize Ask2-Oh51Ao2 and Ask2-Oh545o2, respectively, were used for AK assays, which were performed as described by Brennecke et al. (1996) with the modifications of Wang et al. (2001b). One unit of activity was defined as the amount of enzyme that catalysed the formation of 1 µmol of aspartyl hydroxamate min–1 at 37 °C. Reactions were performed in a buffer consisting of 20 mM TRIS–HCl, 1 mM DTT, 3% (v/v) glycerol, 8 mM MgSO4, 20 mM ATP, and 480 mM hydroxylamine, pH 7.4. To measure inhibition of AK activity by lysine, reaction rates using 50 mM Asp substrate were measured in the presence of 5 µM to 10 mM L-lysine. The protein concentrations were determined as in Bradford (1976), using a reagent supplied by Bio-Rad (Hercules, CA).
Nucleotide and amino acid sequence analysis
Amino acid sequence alignment was performed using the ClustalW program from European Bioinformatic Institute (http://www2.ebi.ac.uk/clustalw/). The protein subdomain analysis was determined with the Simple Modular Architecture Research Tool (SMART) software at the site http://smart.embl-heidelberg.de/ (Schultz et al., 1998). The DNA sequence alignment for contig construction and routine DNA sequence analysis including translation and restriction analysis was conducted using a PC version of the Sequencher program (Gene Codes Corporation, Ann Arbor, MI).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Cloning of monofunctional aspartate kinase genes and their sequence analysis
In previous studies, QTLs influencing the level of free amino acids in maize endosperm, including lysine and other products of the AK pathway, had been identified (Wang and Larkins, 2001). One QTL on the long arm of chromosome 2 was closely linked with the map position of the ask2 locus (Wang et al., 2001b). To isolate the Ask2 gene, advantage was taken of the maize EST database (http://www.mdb.iastate.edu). A lysine-sensitive AK from Arabidopsis (Tang et al., 1997) was used to BLAST search the database, and this led to the identification of several ESTs with a significant degree of sequence identity. After sequencing, the clones were aligned into three contigs. This result suggested there could be three monofunctional AK genes in the maize genome, similar to the number in Arabidopsis (Frankard et al., 1997; Tang et al., 1997; Yoshioka et al., 2001).
The Ask1 and Ask2 coding sequences were cloned using RNA from the B73 inbred. The Ask1-B73 cDNA was obtained based on the maize EST, AW066359 [GenBank] , which contains 581 amino acids, including a predicted transit peptide region (see Materials and methods). The Ask2-B73 cDNA was obtained based on the nucleic acid sequence of the AW282411 [GenBank] EST. Using PCR with a degenerate primer for the conserved Lys-Phe-Gly-Gly (KFGG) motif and a primer from the 3' end of the AW282411 [GenBank] sequence, most of the coding region of the Ask2-B73 gene was cloned. 5'-RACE was only able to extend the NH2-terminal region by 43 nucleotides, and part of the putative transit peptide-encoding sequence appeared to be missing. However, because the transit peptide is absent from the mature protein, it was not necessary for comparison of enzymatic activity. Several EST clones, AI600499 [GenBank] , AI629668 [GenBank] , AI782896 [GenBank] , and AI901511 [GenBank] fell into a different contig, but they all have disrupted open reading frames and so they were not characterized further.
To investigate the nature of maize monofunctional AK genes, their deduced amino acid sequences were compared with those of well-characterized genes from other plant species and the lysC gene of E. coli. The amino acid sequence alignment of the B73 Ask1 and Ask2 cDNAs showed that the two maize genes share a high degree of identity with each other and similar genes of other species (Fig. 1). As expected, the polypeptides do not contain a COOH-terminal region encoding homoserine dehydrogenase, which is found in bifunctional AK-HSDH enzymes (Muehlbauer et al., 1994b). Like other plant AK genes, the maize sequences encode a predicted NH2-terminal transit peptide region, suggesting the precursor proteins are targeted to plastids. The putative enzymatic active site, the KFGG motif, is present in each AK protein (Tang et al., 1997), further indicating the close relationship of these enzymes. By analysing the maize ASK1 and ASK2 amino acid sequences with SMART software (Schultz et al., 1998; Letunic et al., 2002), several conserved motifs were found. These include the amino acid kinase domain and two tandem ACT domains (Fig. 1), which bind specifically to a particular amino acid leading to regulation of the linked enzyme. In this case, they bind lysine and regulate aspartate kinase activity (http://pfam.wustl.edu/cgi-bin/getdesc?name=ACT).
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K) and 512 (Ask2 maps to a QTL that influences the free amino acid composition of the endosperm
To determine if either Ask gene corresponds to the ask2 locus, it was first determined if Ask1 and Ask2 are single copy sequences. Following restriction endonuclease digestion, DNA from Oh545o2 and Oh51Ao2 was hybridized to the 3'-UTR of the Ask1 and Ask2 genes in Southern blots. The Ask1 probe detected a single, strongly hybridizing band as well as one or two weakly-hybridizing sequences (Fig. 2A). These weaker signals might reflect cross-hybridization with related gene sequences, or perhaps non-specific hybridization. The Ask2 gene showed only a single hybridizing DNA band in both genotypes, indicating a single copy gene sequence (Fig. 2B).
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To establish if either of the Ask genes mapped to the QTL region at the ask2 locus, a pair of gene specific primers for the Ask1 and Ask2 alleles of Oh545 and Oh51A were designed based on their 3'-UTRs. The primers were demonstrated to amplify the appropriate sequences in PCR reactions containing DNA from these inbreds. Subsequently, they were used to analyse the genomes of 76 recombinant inbred lines (F6 generation) from a cross between Oh545o2 and Oh51Ao2. The SSR markers, bnlg1138, bnlg1329, and bnlg1633, which flank the QTL on the long arm of chromosome 2 (Wang and Larkins, 2001), were used to genotype the recombinant inbreds. Linkage analysis was conducted between the SSR markers and the Ask1 and Ask2 genes. Using the MapManager program, it was possible to place the Ask2 gene within 5.1 cM of the bnlg1138 locus (Fig. 2C). The Ask1 gene showed no linkage with any of the SSR markers. The order of the three SSR markers on chromosome 2 was found to be bnlg1138-bnlg1329-bnlg1633, rather than bnlg1138-bnlg1633-bnlg1329, as described in our previous study (Wang et al., 2001a).
There is one amino acid difference in the Ask2 enzymes of Oh545o2 and Oh51Ao2
Based on the identification of Ask2 as the gene corresponding to the ask2 locus, this sequence was used to clone the alleles from Oh545o2 and Oh51Ao2 by RT-PCR. Comparison of the amino acid sequences deduced from the cDNAs showed near identity with the protein encoded by the B73 gene. There were only two differences among the three Ask2 alleles. One is at position 125: lysine is found in Ask2-Oh51Ao2 and Ask2-Oh545o2 while arginine occurs in Ask2-B73 (Fig. 1). The other is at position 512 (position noted in Fig. 1 relative to the B73 allele), where leucine is found in the Oh51Ao2 and B73 alleles, and glutamine occurs in the Oh545 allele.
Complementation of the yeast hom3 mutant by the maize Ask2 alleles
To determine if the properties of the Ask2 enzymes in Oh545o2 and Oh51Ao2 vary as a consequence of the single amino acid difference, the yeast hom3/ura3 mutant a3hu was transformed with plasmids expressing the maize Ask2-Oh545o2 and Ask2-Oh51Ao2 sequences. After a first selection on SD medium lacking uracil (Fig. 3A), all the transformants growing on a medium lacking threonine were able to complement the hom3 mutation (Fig. 3B). Interestingly, the growth rate of the yeast a3hu mutant complemented by the Ask2-Oh545o2 gene was much faster than one complemented by the Ask2-Oh51Ao2 gene in a medium lacking threonine and lysine (Fig. 3B, C).
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Ask2-Oh545o2 and Ask2-Oh51Ao2 have similar lysine-sensitivity properties but differ in basal activity
Activity measurements and lysine feedback inhibition assays were performed on protein extracts from the yeast hom3 mutants expressing the Ask2-Oh545o2 and Ask2-Oh51Ao2 coding sequences. These experiments revealed a difference in AK activity for the two enzymes. Figure 4A shows the enzymes encoded by the two alleles have similar lysine inhibition properties, with an I50 of about 80 µM lysine. Interestingly, the enzyme extract from Ask2-Oh545o2 transformants was found to have a higher activity than that from Ask2-Oh51Ao2 transformants. For example, without lysine in the assay, the specific activity of aspartate kinase was 22.2 µmol min–1 mg–1 protein with Ask2-Oh545o2 versus 12.4 µmol min–1 mg–1 protein for Ask2-Oh51Ao2 (Fig. 4A). This difference in specific activity did not appear to be a consequence of variation in the level of the AK enzyme in the yeast extracts, because immunoblotting with antibodies that detected the His-tag on the recombinant proteins revealed similar amounts of enzyme in the AK assays (Fig. 4B).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Evidence is provided suggesting that allelic variation of the Ask2 locus is responsible for the QTL on the long arm of chromosome 2 that influences the content of several essential amino acids derived from the aspartate pathway in maize endosperm. Our previous study suggested that Ask2 is a candidate gene for the QTL on the long arm of chromosome 2 that affects the free amino acid level in maize endosperm (Wang et al., 2001). Consequently, the monofunctional aspartate kinase gene that maps to this region (Fig. 2C) was cloned and its nucleotide sequence determined. Sequence comparisons between three different Ask2 alleles, including those of B73 and the two parental lines, Oh545o2 and Oh51Ao2, showed that the only difference between the two parental alleles is a single amino acid substitution at a position close to the carboxyl-terminus of the gene (Fig. 1).
Interestingly, biochemical characterization following complementation of the yeast hom3 mutant by the Ask2-Oh545o2 and Ask2-Oh51Ao2 alleles showed that the Ask2-Oh545o2 gene encodes an enzyme with higher basal activity in yeast than the Ask2-Oh51Ao2 allele (Fig. 4A). Aspartate kinase is the first key enzyme to direct the flux of aspartate into several essential amino acids, including lysine, threonine, leucine, and isoleucine (Galili, 1995), so the enhanced activity of this enzyme should lead to a larger production of these other amino acids, and this could reasonably explain the profile of free amino acids found in the endosperm of Oh545o2 (Wang and Larkins, 2001). Thus, the Ask2 locus appears to correspond to the QTL on the long arm of chromosome 2. Naturally occurring enzyme variants such as found for maize Ask2 can play important roles in altering the quality of agricultural crops. A similar single amino acid change in a tomato cell wall invertase was shown to increase soluble carbohydrate levels, an important quality trait for this crop (Fridman et al., 2004). Therefore, it is valuable to investigate the allelic diversity represented in breeding stocks as well as close relatives of crop plants to improve quality.
Based on expression in yeast of the Ask2-Oh545o2 and Ask2-Oh51Ao2 alleles, the enzymes have a similar I50 of about 80 µM of lysine (Fig. 4A). This does not agree with our measurements determined from endosperm extracts, which predicted that Ask2-Oh545o2 encodes an enzyme with higher resistance to lysine inhibition (Wang et al., 2001b). This discrepancy can be explained by the following considerations. First, the maize genome contains at least two genes encoding threonine-sensitive, bifunctional aspartate kinase (Muehlbauer et al., 1994b) and three genes encoding lysine-sensitive, monofunctional aspartate kinase, which could distort the effect of a single aspartate kinase after it is partially purified from maize endosperm extracts. For example, the I50 of lysine for the partially purified enzyme from Oh545o2 endosperm is about 0.5 to 0.6 mM (Wang et al., 2001b), which is much higher than the 80 µM determined in yeast extract. This could be caused by the presence of other enzymes, such as the threonine-sensitive isoforms. This problem should have been avoided, because the yeast hom3 mutant produces no aspartate kinase, and all the enzyme activity detected in yeast extracts should originate from the monofunctional aspartate kinase gene. Second, analysis of protein expressed in E. coli showed the I50 was near 400 µM (not shown), consistent with our prior studies; nonetheless, all the available data indicate that the lysine sensitivity of both AK isoforms is virtually identical.
These results suggest a potential novel regulatory mechanism for monofunctional AK in which the carboxy-terminus of the enzyme plays an important role in regulating its basal activity, although we cannot explain how this happens in terms of protein structure. There is a pair of predicted ACT domains in the monofunctional AK sequence that presumably bind to lysine and inhibit the kinase activity (Kikuchi et al., 1999; Chipman and Shaanan, 2001; Viola, 2001). The single amino acid substitution in Ask2-Oh545o2 is close to, but not in, these domains (Fig. 1), and the two maize alleles encode enzymes with seemingly similar lysine inhibition properties (Fig. 4A). Therefore, it is unlikely that this substitution directly affects lysine binding. One possible explanation for the different basal activities of these enzymes is that the carboxy-terminus of AK regulates the interaction between the same or different subunits of monofunctional AK. Previous biochemical characterization of the maize AK complex suggested that the native enzyme may form a hetero-tetramer (Dotson et al., 1989). The interaction among subunits could affect the structure of the holoenzyme and its catalytic activity. However, other unknown mechanisms could also be involved in this regulation. We are continuing to investigate the function of this amino acid change by studying the enzymatic activity of the different Ask2 isoforms to determine if the kinetic properties of the isoforms differ.
Besides the level of enzyme, feedback inhibition of monofunctional AK by lysine has long been believed to play a critical role in the enzyme's regulation, but its molecular mechanism is poorly understood. In this study, enzyme assays from extracts of transformed yeast cultures confirmed that the maize monofunctional AK is lysine sensitive, with an I50 of about 80 µM (Fig. 4A). This is consistent with a previous prediction based solely on biochemical studies (Dotson et al., 1990a). Although two maize lysine-insensitive AK mutants, ask1 and ask2, were identified more than a decade ago (Azevedo et al., 1990; Dotson et al., 1990b; Muehlbauer et al., 1994a), the difficulty of map based cloning in maize prevented investigation of their molecular basis. The ask2 mutation, which results in a strong allele insensitive to lysine inhibition, could be conveniently cloned by RT-PCR based on the gene sequences that have been described. The Ask1 gene does not correspond to the ask1 locus, but with the increased number of maize ESTs available and the approach used in this study, is should be possible to clone the ask1 gene as well.
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Acknowledgements |
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This research was supported by grants from the Department of Energy (DE-FG03-96ER20242) and Pioneer Hi-Bred (to BAL). JALV was supported by CONACYT–Mexico (44092). We wish to express our appreciation to Dr Rudolf Jung at Pioneer Hi-Bred for assistance in the identification of maize AK ESTs. The protein sequence data reported will appear in the GenBank Protein Sequence databases under the accession numbers 881197 (ZmAK1) and 880131 (ZmAK2).
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Footnotes |
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* Present address: The State Key Laboratory of Genetic Engineering, The School of Life Sciences, Fudan University, Shanghai 200433, China.
Present address: Facultad de Ciencias Químico-Biológicas, Universidad Autónoma de Sinaloa, 80000 Culiacán Sin, Mexico.
Present address: Université Paris-Sud, Institut de Biotechnologie des Plantes, UMR 8618, F-91405, Orsay, France.
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Abbreviations |
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AK, aspartate kinase; DHDPS, dihydropicolinate synthase; HSDH, homoserine dehydrogenase.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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