Structure and regulation of the Arabidopsis thaliana allene oxide synthase gene

Allene oxide synthase (AOS) is encoded by a single intronless gene in Arabidopsis thaliana (L.) Heynh. The promoter region of the AOS gene exhibits, in addition to the elements of a minimal promoter and the presence of general enhancers, cis-elements that, in other promoters, are responsible for stress- and ethylene-responsiveness. Arabidopsis thaliana and Nicotiana tabacum L. were transformed with a chimaeric gene consisting of a 1.9-kb 5′-upstream sequence and the first 95 nucleotides of the AOS coding sequence translationally fused to uid A encoding β-glucuronidase (GUS). Using histochemistry, GUS activity was seen in older leaves, in the bases of petioles and in stipules, during the early stages of carpel development, in maturing pollen grains and at the base of elongated filaments, as well as in abscission-zone scars. A role for jasmonates in floral organ abscission is suggested by these findings. Furthermore, the AOS promoter was activated both locally as well as systemically upon wounding. Jasmonic acid, 12-oxophytodienoic acid and coronatine strongly induced GUS activity. This induction remained confined to the treated leaf when agonists were applied locally to a leaf, suggesting that neither jasmonic acid nor 12-oxophytodienoic acid are physiologically relevant components of the systemic wound signal complex. Rather, the data show that jasmonates behave as local response regulators produced at or around the sites of action in response to appropriate triggers of their synthesis. Received: 21 September 1998 / Accepted: 30 December 1998     

Analysis of flavanone 3-hydroxylase in Arabidopsis seedlings. Coordinate regulation with chalcone synthase and chalcone isomerase.

A genomic clone encoding flavanone 3-hydroxylase (F3H) was isolated from Arabidopsis thaliana. The deduced amino acid sequence is 72 to 94% identical to all previously reported F3H proteins. Low-stringency DNA blot analysis indicated that F3H is encoded by a single gene in Arabidopsis. The F3H locus was mapped to the bottom of chromosome 3 and therefore does not correspond to any of the 13 flavonoid-deficient transparent testa mutants for which a map position is known. Analysis of gene expression in etiolated seedlings exposed to white light and in two putative regulatory mutants, ttg and tt8, demonstrated that the Arabidopsis F3H gene is coordinately expressed with chalcone synthase and chalcone isomerases is seedlings, whereas dihydroflavonol reductase expression is controlled by distinct regulatory mechanisms. The F3H gene may represent a pivotal point in the regulation of flavonoid biosynthesis because its expression is coordinated with different subsets of genes in different plant species.     

Characterization of the GGPP synthase gene family in Arabidopsis thaliana

Geranylgeranyl diphosphate (GGPP) is a key precursor of various isoprenoids that have diverse functions in plant metabolism and development. The annotation of the Arabidopsis thaliana genome predicts 12 genes to encode geranylgeranyl diphosphate synthases (GGPPS). In this study we analyzed GGPPS activity as well as the subcellular localization and tissue-specific expression of the entire protein family in A. thaliana. GGPPS2 (At2g18620), GGPPS3 (At2g18640), GGPPS6 (At3g14530), GGPPS7 (At3g14550), GGPPS8 (At3g20160), GGPPS9 (At3g29430), GGPPS10 (At3g32040) and GGPPS11 (At4g36810) showed GGPPS activity in Escherichia coli, similar to activities reported earlier for GGPPS1 (At1g49530) and GGPPS4 (At2g23800) (Zhu et al. in Plant Cell Physiol 38(3):357–361, 1997a; Plant Mol Biol 35(3):331–341, b). GGPPS12 (At4g38460) did not produce GGPP in E. coli. Based on DNA sequence analysis we propose that GGPPS5 (At3g14510) is a pseudogene. GGPPS–GFP (green fluorescent protein) fusion proteins of the ten functional GGPP synthases localized to plastids, mitochondria and the endoplasmic reticulum, with the majority of the enzymes located in plastids. Gene expression analysis using quantitative real time-PCR, GGPPS promoter-GUS (β-glucuronidase) assays and publicly available microarray data revealed a differential spatio-temporal expression of GGPPS genes. The results suggest that plastids and mitochondria are key subcellular compartments for the synthesis of ubiquitous GGPP-derived isoprenoid species. GGPPS11 and GGPPS1 are the major isozymes responsible for their biosynthesis. All remaining paralogs, encoding six plastidial isozymes and two cytosolic isozymes, were expressed in specific tissues and/or at specific developmental stages, suggesting their role in developmentally regulated isoprenoid biosynthesis. Our results show that of the 12 predicted GGPPS encoded in the A. thaliana genome 10 are functional proteins that can synthesize GGPP. Their specific subcellular location and differential expression pattern suggest subfunctionalization in providing GGPP to specific tissues, developmental stages, or metabolic pathways.     

Cardiolipin synthase of Arabidopsis thaliana

Functional expression studies in microorganisms showed that the Arabidopsis thaliana gene At4g04870 represents the cardiolipin synthase (CLS) gene encoding a hydrophobic preprotein of 38 kDa with a cleavable signal peptide for the import into mitochondria. CLS of Arabidopsis over-expressed in Escherichia coli has an alkaline pH optimum, a strict requirement for divalent cations and a distinctly lower K(m) for cytidinediphosphate-diacylglycerol than for phosphatidylglycerol. It displayed a preference for both its substrates esterified with unsaturated acyl groups. Solubilization and purification experiments revealed that the protein requires a defined phospholipid environment, particularly the presence of cardiolipin, to acquire its catalytically active conformation.     

Expression of chalcone synthase and chalcone isomerase proteins in Arabidopsis seedlings

Antibodies have been developed against the first two enzymes of flavonoid biosynthesis in Arabidopsis thaliana. Chalcone synthase (CHS) and chalcone isomerase (CHI) were overexpressed and purified from Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. The recombinant proteins were then used to immunize chickens and the resulting IgY fraction was purified from egg yolks. Immunoblots of crude protein extracts from Arabidopsis seedlings carrying wild-type and null alleles for CHS and CHI showed that the resulting antibody preparations provide useful tools for characterizing expression of the flavonoid pathway at the protein level. An initial analysis of expression patterns in seedlings shows that CHS and CHI proteins are present at high levels during a brief period of early seedling germination that just precedes the transient accumulation of flavonoid end-products.     

Characterization of recombinant Arabidopsis thaliana threonine synthase.

Threonine synthase (TS) catalyses the last step in the biosynthesis of threonine, the pyridoxal 5'-phosphate dependent conversion of L-homoserine phosphate (HSerP) into L-threonine and inorganic phosphate. Recombinant Arabidopsis thaliana TS (aTS) was characterized to compare a higher plant TS with its counterparts from Escherichia coli and yeast. This comparison revealed several unique properties of aTS: (a) aTS is a regulatory enzyme whose activity was increased up to 85-fold by S-adenosyl-L-methionine (SAM) and specifically inhibited by AMP; (b) HSerP analogues shown previously to be potent inhibitors of E. coli TS failed to inhibit aTS; and (c) aTS was a dimer, while the E. coli and yeast enzymes are monomers. The N-terminal region of aTS is essential for its regulatory properties and protects against inhibition by HSerP analogues, as an aTS devoid of 77 N-terminal residues was neither activated by SAM nor inhibited by AMP, but was inhibited by HSerP analogues. The C-terminal region of aTS seems to be involved in dimer formation, as the N-terminally truncated aTS was also found to be a dimer. These conclusions are supported by a multiple amino-acid sequence alignment, which revealed the existence of two TS subfamilies. aTS was classified as a member of subfamily 1 and its N-terminus is at least 35 residues longer than those of any nonplant TS. Monomeric E. coli and yeast TS are members of subfamily 2, characterized by C-termini extending about 50 residues over those of subfamily 1 members. As a first step towards a better understanding of the properties of aTS, the enzyme was crystallized by the sitting drop vapour diffusion method. The crystals diffracted to beyond 0.28 nm resolution and belonged to the space group P222 (unit cell parameters: a = 6.16 nm, b = 10.54 nm, c = 14.63 nm, alpha = beta = gamma = 90 degrees).     

Metabolic regulation of the gene encoding glutamine-dependent asparagine synthetase in Arabidopsis thaliana.

Here, we characterize a cDNA encoding a glutamine-dependent asparagine synthetase (ASN1) from Arabidopsis thaliana and assess the effects of metabolic regulation on ASN1 mRNA levels. Sequence analysis shows that the predicted ASN1 peptide contains a purF-type glutamine-binding domain. Southern blot experiments and cDNA clone analysis suggest that ASN1 is the only gene encoding glutamine-dependent asparagine synthetase in A. thaliana. The ASN1 gene is expressed predominantly in shoot tissues, where light has a negative effect on its mRNA accumulation. This negative effect of light on ASN1 mRNA levels was shown to be mediated, at least in part, via the photoreceptor phytochrome. We also investigated whether light-induced changes in nitrogen to carbon ratios might exert a metabolic regulation of the ASN1 mRNA accumulation. These experiments demonstrated that the accumulation of ASN1 mRNA in dark-grown plants is strongly repressed by the presence of exogenous sucrose. Moreover, this sucrose repression of ASN1 expression can be partially rescued by supplementation with exogenous amino acids such as asparagine, glutamine, and glutamate. These findings suggest that the expression of the ASN1 gene is under the metabolic control of the nitrogen to carbon ratio in cells. This is consistent with the fact that asparagine, synthesized by the ASN1 gene product, is a favored compound for nitrogen storage and nitrogen transport in dark-grown plants. We have put forth a working model suggesting that when nitrogen to carbon ratios are high, the gene product of ASN1 functions to re-direct the flow of nitrogen into asparagine, which acts as a shunt for storage and/or long-distance transport of nitrogen.     

Biotin synthase from Arabidopsis thaliana. cDNA isolation and characterization of gene expression.

The full-length BIO2 cDNA from Arabidopsis thaliana was isolated using an expressed sequence tag that was homologous to the Escherichia coli biotin synthase gene (BioB). Comparisons of the deduced amino acid sequence from BIO2 with bacterial and yeast biotin synthase homologs revealed a high degree of sequence similarity. The amino terminus of the predicted BIO2 protein contains a stretch of hydrophobic residues similar in composition to transit peptide sequences. BIO2 is a single-copy nuclear gene in Arabidopsis that is expressed at high levels in the tissues of immature plants. Expression of BIO2 was higher in the light relative to dark and was induced 5-fold during biotin-limited conditions. These results demonstrate that expression of at least one gene in this pathway is regulated in response to developmental, environmental, and bio-chemical stimuli.     

Diverse selective modes among orthologs/paralogs of the chalcone synthase (Chs) gene family of Arabidopsis thaliana and its relative A. halleri ssp. gemmifera

As a model system, Arabidopsis thaliana and its wild relatives have played an important role in the study of genomics and evolution in plants. In this study, we examined the genetic diversity of the chalcone synthase (Chs) gene, which encodes a key enzyme of the flavonoid pathway and is located on chromosome five, as well as two Chs-like genes on the first and fourth chromosomes of Arabidopsis. The objectives of the study are to determine if natural selection operates differentially on the paralogs of the Chs gene family in A. thaliana and Arabidopsis halleri ssp. gemmifera. The mode of selection was inferred from Tajima's D values from noncoding and coding regions, as well as from the ratio of nonsynonymous to synonymous substitutions. Both McDonald-Kreitman and HKA tests revealed the effects of selection on the allelic distribution, except for the chromosome 1 paralog in ssp. gemmifera. The Chs gene on chromosome 5 was under purifying selection in both species. Significant, negative Tajima's D values at synonymous sites and positive Fay and Wu's H values within coding region, plus reduced genetic variability in introns, indicated effects of background selection in shaping the evolution of this gene region in A. thaliana. The Chs paralog on chromosome 1 was under positive selection in A. thaliana, while interspecific introgression and balancing selection determined the fates of the paralog and resulted in high heterogeneity in ssp. gemmifera. Local adaptation differentiated populations of Japan and China at the locus. In contrast, the other Chs-paralog of chromosome 4 was shaped by purifying selection in A. thaliana, while under positive selection in ssp. gemmifera, as indicated by dn/ds>1. Moreover, these contrasting patterns of selection have likely resulted in functional divergence in Arabidopsis, as indicated by radical amino acid substitutions at the chalcone synthase/stilbene synthase motif of the Chs genes. Unlike previous studies of the evolutionary history of A. thaliana, the high levels of genetic diversity in most gene regions of Chs paralogs and nonsignificant Tajima's D in the intron sequences of the Chs gene family in A. thaliana did not reflect the effects of a recent demographic expansion.     

Isolation and expression analysis of the isopropylmalate synthase gene family of Arabidopsis thaliana

Isopropylmalate synthase (IPMS) is the first enzyme in the leucine biosynthetic pathway. It is the branch point in the biosynthesis of leucine and the other branched-chain amino acids. IPMS is also regulated by negative feedback inhibition by the end-product leucine. There are four highly homologous loci within the Arabidopsis thaliana genome, which contain sequences that code for IPMS. Through library screening and RT-PCR the expression patterns of three of these loci namely IMS1, IMS2, and IMS3 have been isolated and then characterized. cDNAs of IMS2 and IMS3 lacking the 5' chloroplast leader sequence were able to complement a leucine auxotroph of E. coli.     

An allelic series for the chalcone synthase locus in Arabidopsis

Five new alleles of the Arabidopsis chalcone synthase (CHS) locus, tt4, have been characterized at the gene, protein, and end product levels as a genetic approach to understanding structure-function relationships in a key enzyme of plant secondary metabolism. Together with two previously described mutants, these tt4 lines represent one of the first allelic series for a central enzyme of the flavonoid pathway and include both null alleles and alleles with leaky, apparently temperature-sensitive, phenotypes. A variety of effects on accumulation of CHS protein and flavonoid glycosides were observed among these lines, including alterations in the apparent stability and activity of the enzyme. Assembly of the CHS homodimer also appeared to be impacted in several cases. A three-dimensional model of the Arabidopsis CHS protein, based on the recently determined structure for alfalfa CHS, predicts significant effects on protein structure or folding for several of the mutations. This allelic series should provide a useful genetic resource for ongoing studies of flavonoid enzyme structure, function, and subcellular organization.