Cloning of a cDNA encoding ATP sulfurylase from Arabidopsis thaliana by functional expression in Saccharomyces cerevisiae.

ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of a cDNA encoding ATP sulfurylase (APS1) from Arabidopsis thaliana. APS1 was isolated by its ability to alleviate the methionine requirement of an ATP sulfurylase mutant strain of Saccharomyces cerevisiae (yeast). Expression of APS1 correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1748-bp cDNA with an open reading frame predicted to encode a 463-amino acid, 51,372-D protein. The predicted amino acid sequence of APS1 is similar to ATP sulfurylase of S. cerevisiae, with which it is 25% identical. Two lines of evidence indicate that APS1 encodes a chloroplast form of ATP sulfurylase. Its predicted amino-terminal sequence resembles a chloroplast transit peptide; and the APS1 polypeptide, synthesized in vitro, is capable of entering isolated intact chloroplasts. Several genomic DNA fragments that hybridize with the APS1 probe were identified. The APS1 cDNA hybridizes to three species of mRNA in leaves (1.85, 1.60, and 1.20 kb) and to a single species of mRNA in roots (1.85 kb).     

Identification of the gene encoding homoserine kinase from Arabidopsis thaliana and characterization of the recombinant enzyme derived from the gene

Homoserine kinase (EC 2.7.1.39) catalyzes the formation of O-phospho-l-homoserine, a branch point intermediate in the pathways for Met and Thr in plants. A genomic open reading frame located on the top arm of chromosome II and a corresponding cDNA have been identified from Arabidopsis thaliana that encode homoserine kinase. The HSK gene is composed of an 1113-bp continuous open reading frame that could produce a 38-kDa protein. The gene product has homology with homoserine kinase from bacteria and fungi. It contains a conserved motif, known as GHMP, found in a group of ATP-dependent metabolite kinases and thought to comprise the ATP binding site. The amino-terminal 50 amino acids of the HSK protein show features of a transit peptide for localization to plastids. Genomic blot analysis revealed that there is a single locus in A. thaliana to which the HSK cDNA hybridizes. The HSK protein expressed as a His-tagged construct in Escherichia coli shows a specific activity in an l-homoserine-dependent ADP synthesis assay of 3.09 +/- 0.25 micromol min(-1) mg(-1) protein at pH 8.5 and 37 degrees C. The apparent K(m) values are 0.40 mM for l-homoserine and 0.32 mM for Mg-ATP. Other hydroxylated compounds are not used as substrates. The enzyme requires 40 mM K(+) and 3 mM Mg(2+) for activity. It has an unusually high temperature optimum, yet it is very unstable, losing more than 80% of its activity after a single cycle of freeze-thawing. The HSK enzyme shows no significant regulation by amino acids in vitro.     

Cloning of a temperature-regulated gene encoding a chloroplast omega-3 desaturase from Arabidopsis thaliana.

Previous genetic evidence suggested that the fad8 and fad7 genes of Arabidopsis thaliana encode chloroplast membrane-associated omega-3 desaturases. A putative fad8 cDNA was isolated by heterologous hybridization using a gene encoding an endoplasmic reticulum-localized omega-3 desaturase (fad3) as a probe. The cDNA encodes a protein of 435 amino acid residues with a molecular mass of 50,134 D. Constitutive expression of the cDNA in transgenic plants of a fad7 mutant resulted in genetic complementation of the mutation, indicating that the fad7 and fad8 gene products are functionally equivalent. Expression of the fad8 cDNA in transgenic plants often resulted in the co-suppression of both the endogenous fad7 and fad8 genes in spite of the fact that these two genes share only about 75% nucleotide identity. In contrast to all other known plant desaturases, including fad7, the steady-state level of fad8 mRNA is strongly increased in plants grown at low temperature. This suggests that the role of fad8 is to provide increased omega-3 desaturase activity in plants that are exposed to low growth temperature. The fad8-1 mutation created a premature stop codon 149 amino acids from the amino-terminal end of the fad8 open reading frame, suggesting that this mutation results in a complete loss of fad8 activity.     

Structural and functional insights into (S)-ureidoglycine aminohydrolase, key enzyme of purine catabolism in Arabidopsis thaliana

The ureide pathway has recently been identified as the metabolic route of purine catabolism in plants and some bacteria. In this pathway, uric acid, which is a major product of the early stage of purine catabolism, is degraded into glyoxylate and ammonia via stepwise reactions of seven different enzymes. Therefore, the pathway has a possible physiological role in mobilization of purine ring nitrogen for further assimilation. (S)-Ureidoglycine aminohydrolase enzyme converts (S)-ureidoglycine into (S)-ureidoglycolate and ammonia, providing the final substrate to the pathway. Here, we report a structural and functional analysis of this enzyme from Arabidopsis thaliana (AtUGlyAH). The crystal structure of AtUGlyAH in the ligand-free form shows a monomer structure in the bicupin fold of the β-barrel and an octameric functional unit as well as a Mn(2+) ion binding site. The structure of AtUGlyAH in complex with (S)-ureidoglycine revealed that the Mn(2+) ion acts as a molecular anchor to bind (S)-ureidoglycine, and its binding mode dictates the enantioselectivity of the reaction. Further kinetic analysis characterized the functional roles of the active site residues, including the Mn(2+) ion binding site and residues in the vicinity of (S)-ureidoglycine. These analyses provide molecular insights into the structure of the enzyme and its possible catalytic mechanism.     

Identification of the Arabidopsis thaliana flavonoid 3'-hydroxylase gene and functional expression of the encoded P450 enzyme

The phenylpropanoid pathway results in the synthesis of thousands of compounds, including flavonoids like flavonols, anthocyanidins and tannins. In Arabidopsis thaliana, the lack of tannins in the seed coat (testa) causes the transparent testa (tt) phenotype. In the present study, we identified the gene responsible for the tt7 mutation. We show that TT7 encodes the enzyme flavonoid 3'-hydroxylase (F3'H), and demonstrate that this P450-dependent monooxygenase has F3'H activity. The availability of the AtF3'H gene and promoter sequence will allow us to study the coregulation of a complete set of flavonol and anthocyanidin biosynthesis genes in A. thaliana, and makes in vitro synthesis of hydroxylated flavonoids more feasible.     

Floral expression of a gene encoding an E2-relatedubiquitin-conjugating protein from Arabidopsis thaliana

An Arabidopsis thaliana gene (UBC6) encoding a homologue to ubiquitin-conjugating enzymes has been isolated which is capable of encoding a protein of 183 amino acids of ca. 21 kDa. Northern analysis indicates that the gene is expressed in flowers, seeds and, to a somewhat lesser extent, in 10-day seedlings but not in mature leaves, callus and pre-flowering plants. This pattern of expression is confirmed using transgenic Arabidopsis plants containing a UBC6 promoter-GUS gene fusion construct. These plants displey GUS activity in mature anthers prior to dehiscence, in developing embryos, sepals and the style after pollination.     

Identification of genes encoding adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis thaliana

The initial step in the de novo biosynthesis of cytokinin in higher plants is the formation of isopentenyladenosine 5'-monophosphate (iPMP) from AMP and dimethylallylpyrophosphate (DMAPP), which is catalyzed by adenylate isopentenyltransferase (IPT). Although cytokinin is an essential hormone for growth and development, the nature of the enzyme for its biosynthesis in higher plants has not been identified. Herein, we describe the molecular cloning and biochemical identification of IPTs from Arabidopsis thaliana. Eight cDNAs encoding putative IPT, designated as AtIPT1 to AtIPT8, were picked up from A. thaliana. The Escherichia coli transformants expressing the recombinant proteins excreted cytokinin species into the culture medium except for that expressing AtIPT2 that is a putative tRNA IPT. A purified recombinant AtIPT1 catalyzed the formation of iPMP from DMAPP and AMP. These results indicate that the small multigene family contains both types of isopentenyltransferase, which could synthesize cytokinin and mature tRNA.     

Cloning and expression of an Arabidopsis thaliana cDNA encoding a monofunctional aspartate kinase homologous to the lysine-sensitive enzyme of Escherichia coli

As in many bacterial species, the first enzymatic reaction of the aspartate-family pathway in plants is mediated by several isozymes of aspartate kinase (AK) that are subject to feedback inhibition by the end-product amino acids lysine or threonine. So far, only cDNAs and genes encoding threonine-sensitive AKs have been cloned from plants. These were all shown to encode polypeptides containing two linked activities, namely AK and homoserine dehydrogenase (HSD), similar to the Escherichia coli thrA gene encoding a threonine-sensitive bifunctional AK/HSD isozyme. In the present report, we describe the cloning of a new Arabidopsis thaliana cDNA that is relatively highly homologous to the E. coli lysC gene encoding the lysine-sensitive AK isozyme. Moreover, similar to the bacterial lysine-sensitive AK, the polypeptide encoded by the present cDNA is monofunctional and does not contain an HSD domain. These observations imply that our cloned cDNA encodes a lysine-sensitive AK. Southern blot hybridization detected a single gene highly homologous to the present cDNA, plus an additional much less homologous gene. This was confirmed by the independent cloning of an additional Arabidopsis cDNA encoding a lysine-sensitive AK (see accompanying paper). Northern blot analysis suggested that the gene encoding this monofunctional AK cDNA is abundantly expressed in most if not all tissues of Arabidopsis.     

Genomic and functional characterization of the oas gene family encoding O-acetylserine (thiol) lyases, enzymes catalyzing the final step in cysteine biosynthesis in Arabidopsis thaliana

The final step of cysteine biosynthesis in plants is catalyzed by O-acetylserine (thiol) lyase (OAS-TL), which occurs as several isoforms found in the cytosol, the plastids and the mitochondria. Genomic DNA blot hybridization and isolation of genomic clones indicate single copy genes (oasA1, oasA2, oasB and oasC) that encode the activities of OAS-TL A, B and C found in separate subcellular compartments in the model plant Arabidopsis thaliana. Sequence analysis reveals that the newly discovered oasA2 gene represents a pseudogene that is still transcribed, but is not functionally translated. The comparison of gene structures suggests that oasA1/oasA2 and oasB/oasC are closely related and may be derived from a common ancestor by subsequent duplications. OAS-TL A, B and C were overexpressed in an Escherichia coli mutant lacking cysteine synthesis and exhibited bifunctional OAS-TL and beta-cyanoalanine synthase (CAS) activities. However, all three proteins represent true OAS-TLs according to kinetic analysis and are unlikely to function in cyanide detoxification or secondary metabolism. In addition, it was demonstrated that the mitochondrial OAS-TL C exhibits in vivo protein-protein interaction capabilities with respect to cysteine synthase complex formation similar to cytosolic OAS-TL A and plastid OAS-TL B. Multiple database accessions for each of the A. thaliana OAS-TL isoforms can thus be attributed to a specified number of oas genes to which functionally defined gene products are assigned, and which are responsible for compartment-specific cysteine synthesis.     

Cloning of Arabidopsis thaliana phosphatidylinositol synthase and functional expression in the yeast pis mutant

It is believed that phosphatidylinositol (PI) metabolism plays a central role in signalling pathways in both animals and higher plants. PI is synthesized from CDP-diacylglycerol (CDP-DG) and myo-inositol by phosphatidylinositol synthase (PI synthase, EC 2.7.8.11). Here we report the identification of a plant cDNA (AtPIS1) encoding a 26 kDa PI synthase from Arabidopsis thaliana. The plant enzyme as deduced from its cDNA sequence shares 35–41% identical amino acids with PI synthases from Saccharomyces cerevisiae and mammals. AtPIS1 functionally complements a mutant of S. cerevisiae with a lesion in PI synthase, and recombinant AtPIS1 protein present in yeast membranes strongly depends on the two principal substrates, myo-inositol and CDP-DG, and requires Mg²⁺ ions for full activity.     

Identification and characterization of the Arabidopsis gene encoding the tetrapyrrole biosynthesis enzyme uroporphyrinogen III synthase

UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo.     

Cloning and expression analyses of AtMRP4, a novel MRP-like gene from Arabidopsis thaliana

In all organisms glutathione-conjugate transporters (GS-X pumps) mediate the detoxification of a number of xenobiotics by removing them from the cytosol. In addition, GS-X pumps appear to play a role in the processing of endogenous compounds. We have isolated a novel genomic clone from Arabidopsis thaliana that encodes a putative GS-X pump, AtMRP4, which is part of a recently defined gene family. The derived amino acid sequence shares high levels of similarity (55–63%) with human, yeast, and other Arabidopsis homologues. The expression of the different members of the AtMRP gene family in Arabidopsis cell suspensions after treatment with chemicals that modify glutathione metabolism (compounds that induce different types of stress and that act as herbicide antidotes – safeners – in monocotyledonous species) revealed that the members of this gene family are differentially regulated.     

Cloning and functional expression of AtCOQ3, the Arabidopsis homologue of the yeast COQ3 gene, encoding a methyltransferase from plant mitochondria involved in ubiquinone biosynthesis

A mutant of Saccharomyces cerevisiae deleted for the COQ3 gene was constructed. COQ3 encodes a 3,4-dihydroxy-5-hexaprenylbenzoate (DHHB) methyltransferase that catalyses the fourth step in the biosynthesis of ubiquinone from p-hydroxybenzoic acid. A full length cDNA encoding a homologue of DHHB-methyltransferase was cloned from an Arabidopsis thaliana cDNA library by functional complementation of a yeast coq3 deletion mutant. The Arabidopsis thaliana cDNA (AtCOQ3) was able to restore the respiration ability and ubiquinone synthesis of the mutant. The product of the 1372 bp cDNA contained 322 amino acids and had a molecular mass of 35 360 Da. The predicted amino acid sequence contained all consensus regions for S-adenosyl methionine methyltransferases and presented 26% identity with Saccharomyces cerevisiae DHHB-methyltransferase and 38% identity with the rat protein, as well as with a bacterial (Escherichia coli and Salmonella typhimurium) methyltransferase encoded by the UBIG gene. Southern analysis showed that the Arabidopsis thaliana enzyme was encoded by a single nuclear gene. The NH₂-terminal part of the cDNA product contained features consistent with a putative mitochondrial transit sequence. The cDNA in Escherichia coli was overexpressed and antibodies were raised against the recombinant protein. Western blot analysis of Arabidopsis thaliana and pea protein extracts indicated that the AtCOQ3 gene product is localized within mitochondrial membranes. This result suggests that at least this step of ubiquinone synthesis takes place in mitochondria.     

Structural and functional analyses of Arabidopsis thaliana chlorophyll a/b-binding protein (cab) promoters

Arabidopsis thaliana carries three functional copies of the chlorophyll a/b-binding protein (cab) gene which code for an identical mature protein. DNA sequence comparison of all three cab promoters indicated that cab2 and cab3 are more closely related compared to cab1. Although the highest degree of homology was found between the TATA box and -256 of cab3 promoter, suggesting that this region plays a major role in promoter function, this promoter regions are only 47% homologous. To study whether these promoters are regulated by identical cis-acting regulatory elements, the promoters were mutated by progressive deletions and the effects on the promoter activity were measured in either transformed plants or cultured cells. It was found that the minimum sequence necessary for the light-dependent tissue-specific promoter activity of the cab3 is the 89 bp DNA fragment (between -74 and -164) at the region of the TATA and the CCAAT boxes. However, an additional 45 bp DNA fragment (between -164 and -209) upstream of the CCAAT box was necessary for the full promoter activity in the leaves. The regulatory element in the 45 bp region appears to be a positive regulator or enhancer which is specific to photosynthetic cells, since the region did not enhance the promoter activity in cultured cells. This region contains an octamer, TGCCACGT (cab2) or TGCCACAT (cab3), which is similar to the previously identified element, TGACACGT from Arabidopsis cab1 promoter. The upstream regions of the cab promoters appear to contain additional elements which are functionally distinct in each promoter since the upstream region of cab1 activated a non-functional nos promoter whereas that of cab3 did not.     

The ubiquitin-activating enzyme (E1) gene family in Arabidopsis thaliana

Conjugation of multiple ubiquitins serves as a committed step in the degradation of a variety of intracellular eukaryotic proteins by the 26S proteasome. Conjugates are formed via a three-enzyme cascade; the initial step requires ubiquitin-activating enzyme (E1), which couples ubiquitin activation to ATP hydrolysis. Previously, we showed that many higher plants contain multiple E1 proteins and described several E1 genes from wheat. To facilitate understanding of the roles of the different plant E1s, we characterized the E1 gene and protein family from Arabidopsis thaliana . Arabidopsis E1s are encoded by two genes ( AtUBA1 and AtUBA2 ) that synthesize approximately 123-kDa proteins with 81% amino acid sequence identity to each other and 44–75% sequence identity with confirmed E1s from other organisms. Like other E1 proteins, AtUBA1 and 2 contain a cysteine residue in the putative active site for forming the ubiquitin thiol-ester intermediate. Enzymatic analysis of the corresponding proteins expressed in Escherichia coli demonstrated that both proteins activate ubiquitin in an ATP-dependent reaction and transfer the activated ubiquitin to a variety of Arabidopsis E2s with near equal specificity. Expression studies by quantitative RT-PCR and histochemistry with transgenic plants containing AtUBA promoter-β-glucuronidase-coding region fusions showed that the AtUBA1 and 2 genes are co-expressed in most, if not all, Arabidopsis tissues and cells. Collectively, the data indicate that E1 proteins, and presumably the rest of the ubiquitin pathway, are present throughout Arabidopsis . They also show that the AtUBA1 and 2 genes are not differentially expressed nor do they encode E1s with dramatically distinct enzymatic properties.