Gene_locus Report for: arath-CXE12

Arabidopsis thaliana contains multiple carboxyesterases (AtCXEs) with activities toward xenobiotics, including herbicide esters that are activated to their phytotoxic acids upon hydrolysis. On the basis of their susceptibility to inhibition by organophosphates, these AtCXEs are all serine hydrolases. Using a trifunctional probe bearing a fluorophosphonate together with biotin and rhodamine to facilitate detection and recovery, four dominant serine hydrolases were identified in the proteome of Arabidopsis. Using a combination of protein purification, capture with the trifunctional probe and proteomics, one of these hydrolases, AtCXE12, was shown to be the major carboxyesterase responsible for hydrolyzing the pro-herbicide methyl-2,4-dichlorophenoxyacetate (2,4-D-methyl) to the phytotoxic acid 2,4-dichlorophenoxyacetic acid. Recombinant expression of the other identified hydrolases showed that AtCXE12 was unique in hydrolyzing 2,4-D-methyl. To determine the importance of AtCXE12 in herbicide metabolism and efficacy, the respective tDNA knock-out (atcxe12) plants were characterized and shown to lack expression of AtCXE12 and have greatly reduced levels of 2,4-D-methyl-hydrolyzing activity. Young atcxe12 seedlings were less sensitive than wild type plants to 2,4-D-methyl, confirming a role for the enzyme in herbicide bioactivation in Arabidopsis.

Carboxylesterases hydrolyze esters of short-chain fatty acids and have roles in animals ranging from signal transduction to xenobiotic detoxification. In plants, however, little is known of their roles. We have systematically mined the genome from the model plant Arabidopsis thaliana for carboxylesterase genes and studied their distribution in the genome and expression profile across a range of tissues. Twenty carboxylesterase genes (AtCXE) were identified. The AtCXE family shares conserved sequence motifs and secondary structure characteristics with carboxylesterases and other members of the larger alpha/beta hydrolase fold superfamily of enzymes. Phylogenetic analysis of the AtCXE genes together with other plant carboxylesterases distinguishes seven distinct clades, with an Arabidopsis thaliana gene represented in six of the seven clades. The AtCXE genes are widely distributed across the genome (present in four of five chromosomes), with the exception of three clusters of tandemly duplicated genes. Of the interchromosomal duplication events, two have been mediated through newly identified partial chromosomal duplication events that also include other genes surrounding the AtCXE loci. Eighteen of the 20 AtCXE genes are expressed over a broad range of tissues, while the remaining 2 (unrelated) genes are expressed only in the flowers and siliques. Finally, hypotheses for the functional roles of the AtCXE family members are presented based on the phylogenetic relationships with other plant carboxylesterases of known function, their expression profile, and knowledge of likely esterase substrates found in plants.

Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.

Arabidopsis thaliana contains multiple carboxyesterases (AtCXEs) with activities toward xenobiotics, including herbicide esters that are activated to their phytotoxic acids upon hydrolysis. On the basis of their susceptibility to inhibition by organophosphates, these AtCXEs are all serine hydrolases. Using a trifunctional probe bearing a fluorophosphonate together with biotin and rhodamine to facilitate detection and recovery, four dominant serine hydrolases were identified in the proteome of Arabidopsis. Using a combination of protein purification, capture with the trifunctional probe and proteomics, one of these hydrolases, AtCXE12, was shown to be the major carboxyesterase responsible for hydrolyzing the pro-herbicide methyl-2,4-dichlorophenoxyacetate (2,4-D-methyl) to the phytotoxic acid 2,4-dichlorophenoxyacetic acid. Recombinant expression of the other identified hydrolases showed that AtCXE12 was unique in hydrolyzing 2,4-D-methyl. To determine the importance of AtCXE12 in herbicide metabolism and efficacy, the respective tDNA knock-out (atcxe12) plants were characterized and shown to lack expression of AtCXE12 and have greatly reduced levels of 2,4-D-methyl-hydrolyzing activity. Young atcxe12 seedlings were less sensitive than wild type plants to 2,4-D-methyl, confirming a role for the enzyme in herbicide bioactivation in Arabidopsis.

Carboxylesterases hydrolyze esters of short-chain fatty acids and have roles in animals ranging from signal transduction to xenobiotic detoxification. In plants, however, little is known of their roles. We have systematically mined the genome from the model plant Arabidopsis thaliana for carboxylesterase genes and studied their distribution in the genome and expression profile across a range of tissues. Twenty carboxylesterase genes (AtCXE) were identified. The AtCXE family shares conserved sequence motifs and secondary structure characteristics with carboxylesterases and other members of the larger alpha/beta hydrolase fold superfamily of enzymes. Phylogenetic analysis of the AtCXE genes together with other plant carboxylesterases distinguishes seven distinct clades, with an Arabidopsis thaliana gene represented in six of the seven clades. The AtCXE genes are widely distributed across the genome (present in four of five chromosomes), with the exception of three clusters of tandemly duplicated genes. Of the interchromosomal duplication events, two have been mediated through newly identified partial chromosomal duplication events that also include other genes surrounding the AtCXE loci. Eighteen of the 20 AtCXE genes are expressed over a broad range of tissues, while the remaining 2 (unrelated) genes are expressed only in the flowers and siliques. Finally, hypotheses for the functional roles of the AtCXE family members are presented based on the phylogenetic relationships with other plant carboxylesterases of known function, their expression profile, and knowledge of likely esterase substrates found in plants.

Functional analysis of a genome requires accurate gene structure information and a complete gene inventory. A dual experimental strategy was used to verify and correct the initial genome sequence annotation of the reference plant Arabidopsis. Sequencing full-length cDNAs and hybridizations using RNA populations from various tissues to a set of high-density oligonucleotide arrays spanning the entire genome allowed the accurate annotation of thousands of gene structures. We identified 5817 novel transcription units, including a substantial amount of antisense gene transcription, and 40 genes within the genetically defined centromeres. This approach resulted in completion of approximately 30% of the Arabidopsis ORFeome as a resource for global functional experimentation of the plant proteome.

Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.