Thaliana Genome Sequence Research Articles

The gene space of European mistletoe (Viscum album).

European mistletoe (Viscum album) is a hemiparasitic flowering plant that is known for its very special life cycle and extraordinary biochemical properties. Particularly, V. album has an unusual mode of cellular respiration that takes place in the absence of mitochondrial complex I. However, insights into the molecular biology of V. album so far are very limited. Since the genome of V. album is extremely large (estimated 600 times larger than the genome of the model plant Arabidopsis thaliana) it has not been sequenced up to now. We here report sequencing of the V. album gene space (defined as the space including and surrounding genic regions, encompassing coding as well as 5' and 3' non-coding regions). mRNA fractions were isolated from different V. album organs harvested in summer or winter and were analyzed via single-molecule real-time sequencing. We determined sequences of 39 092 distinct open reading frames encoding 32 064 V. album proteins (designated V. album protein space). Our data give new insights into the metabolism and molecular biology of V. album, including the biosynthesis of lectins and viscotoxins. The benefits of the V. album gene space information are demonstrated by re-evaluating mass spectrometry-based data of the V. album mitochondrial proteome, which previously had been evaluated using the A. thaliana genome sequence. Our re-examination allowed the additional identification of nearly 200 mitochondrial proteins, including four proteins related to complex I, which all have a secondary function not related to respiratory electron transport. The V. album gene space sequences are available at the NCBI.

Anno genominis XX: 20 years of Arabidopsis genomics.

Twenty years ago, the Arabidopsis thaliana genome sequence was published. This was an important moment as it was the first sequenced plant genome and explicitly brought plant science into the genomics era. At the time, this was not only an outstanding technological achievement, but it was characterized by a superb global collaboration. The Arabidopsis genome was the seed for plant genomic research. Here, we review the development of numerous resources based on the genome that have enabled discoveries across plant species, which has enhanced our understanding of how plants function and interact with their environments.

Orthologous Revelation between Elaeis guineensis, Arabidopsis thaliana and Solanum lycopersicum

Oil palms are important commodity crop in Malaysia as major contributor to agriculture sector. Thus, the need for better yield production is urgent to accommodate rising local and global demand while reducing the land expansion for oil palm plantation. This can be achieved by identifying the agronomical important traits in oil palm using comparative genomic approach. In this study, gene related to plant height, fruit development and fruit ripening in oil palm were predicted by comparing Elaeis guineensis genome sequence with Arabidopsis thaliana and Solanum lycopersicum genome sequence. The model plant chosen are based on its special attribute such as completely sequenced and fleshy fruit model. The analysis begun with orthology analysis using InParanoid, and SonicParanoid. There are 9,624 orthologous genes identified common among species selected. The reformatted orthologous genes were then annotated with Gene Ontology (GO) using Blast2GO program. The process of annotation include blast with local database (DIAMOND), mapping, annotation and project statistical analysis. 100% of the orthologous gene blasted has sequence of significant similarity but only 61.79% of the sequence has GO assignment. By using the annotated orthologous genes generated, only small amount of gene associated with trait of interest was predicted i.e. gibberellins (GAs) 10 genes, bassinosteroids (BRs) 14 genes, auxin (9 genes), fruit development (8 genes) and fruit ripeing (4 genes). These agronomical important genes could be utilized in genetic engineering and molecular breeding to improve the production of palm oils.

Araport: the Arabidopsis information portal.

The Arabidopsis Information Portal (https://www.araport.org) is a new online resource for plant biology research. It houses the Arabidopsis thaliana genome sequence and associated annotation. It was conceived as a framework that allows the research community to develop and release ‘modules’ that integrate, analyze and visualize Arabidopsis data that may reside at remote sites. The current implementation provides an indexed database of core genomic information. These data are made available through feature-rich web applications that provide search, data mining, and genome browser functionality, and also by bulk download and web services. Araport uses software from the InterMine and JBrowse projects to expose curated data from TAIR, GO, BAR, EBI, UniProt, PubMed and EPIC CoGe. The site also hosts ‘science apps,’ developed as prototypes for community modules that use dynamic web pages to present data obtained on-demand from third-party servers via RESTful web services. Designed for sustainability, the Arabidopsis Information Portal strategy exploits existing scientific computing infrastructure, adopts a practical mixture of data integration technologies and encourages collaborative enhancement of the resource by its user community.

Initial characteristics of RbcX proteins from Arabidopsis thaliana

Form I of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) is composed of eight large (RbcL) and eight small (RbcS) subunits. Assembly of these subunits into a functional holoenzyme requires the assistance of additional assembly factors. One such factor is RbcX, which has been demonstrated to act as a chaperone in the assembly of most cyanobacterial Rubisco complexes expressed in heterologous system established in Escherichia coli cells. Analysis of Arabidopsis thaliana genomic sequence revealed the presence of two genes encoding putative homologues of cyanobacterial RbcX protein: AtRbcX1 (At4G04330) and AtRbcX2 (At5G19855). In general, both RbcX homologues seem to have the same function which is chaperone activity during Rubisco biogenesis. However, detailed analysis revealed slight differences between them. AtRbcX2 is localized in the stromal fraction of chloroplasts whereas AtRbcX1 was found in the insoluble fraction corresponding with thylakoid membranes. Search for putative “partners” using mass spectrometry analysis suggested that apart from binding to RbcL, AtRbcX1 may also interact with β subunit of chloroplast ATP synthase. Quantitative RT-PCR analysis of AtRbcX1 and AtRbcX2 expression under various stress conditions indicated that AtRbcX2 is transcribed at a relatively stable level, while the transcription level of AtRbcX1 varies significantly. In addition, we present the attempts to elucidate the secondary structure of AtRbcX proteins using CD spectroscopy. Presented results are the first known approach to elucidate the role of RbcX proteins in Rubisco assembly in higher plants.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-011-9823-8) contains supplementary material, which is available to authorized users.

Leveraging Genomic Resources of Model Species for the Assessment of Diversity and Phylogeny in Wild and Domesticated Lentil

Advances in comparative genomics have provided significant opportunities for analysis of genetic diversity in species with limited genomic resources, such as the genus Lens. Medicago truncatula expressed sequence tags (ESTs) were aligned with the Arabidopsis thaliana genome sequence to identify conserved exon sequences and splice sites in the ESTs. Conserved primers (CPs) based on M. truncatula EST sequences flanking one or more introns were then designed. A total of 22% of the CPs produced polymerase chain reaction amplicons in lentil and were used to sequence amplicons in 175 wild and 133 domesticated lentil accessions. Analysis of the sequences confirmed that L. nigricans and L. ervoides are well-defined species at the DNA sequence level. Lens culinaris subsp. odemensis, L. culinaris subsp. tomentosus, and L. lamottei may constitute a single taxon pending verification with crossability experiments. Lens culinaris subsp. orientalis is the progenitor of domesticated lentil, L. culinaris subsp. culinaris (as proposed before), but a more specific area of origin can be suggested in southern Turkey. We were also able to detect the divergence, following domestication, of the domesticated gene pool into overlapping large-seeded (megasperma) and small-seeded (microsperma) groups. Lentil domestication led to a loss of genetic diversity of approximately 40%. The approach followed in this research has allowed us to rapidly exploit sequence information from model plant species for the study of genetic diversity of a crop such as lentil with limited genomic resources.

Characterization of Two Novel Aldo–Keto Reductases from Arabidopsis: Expression Patterns, Broad Substrate Specificity, and an Open Active-Site Structure Suggest a Role in Toxicant Metabolism Following Stress

Aldo–keto reductases (AKRs) are widely distributed in nature and play numerous roles in the metabolism of steroids, sugars, and other carbonyls. They have also frequently been implicated in the metabolism of exogenous and endogenous toxicants, including those stimulated by stress. Although the Arabidopsis genome includes at least 21 genes with the AKR signature, very little is known of their functions. In this study, we have screened the Arabidopsis thaliana genomic sequence for genes with significant homology to members of the mammalian AKR1 family and identified four homologues for further study. Following alignment of the predicted protein sequences with representatives from the AKR superfamily, the proteins were ascribed not to the AKR1 family but to the AKR4C subfamily, with the individual designations of AKR4C8, AKR4C9, AKR4C10, and AKR4C11. Expression of two of the genes, AKR4C8 and AKR4C9, has been shown to be coordinately regulated and markedly induced by various forms of stress. The genes have been overexpressed in bacteria, and recombinant proteins have been purified and crystallized. Both enzymes display NADPH-dependent reduction of carbonyl compounds, typical of the superfamily, but will accept a very wide range of substrates, reducing a range of steroids, sugars, and aliphatic and aromatic aldehydes/ketones, although there are distinct differences between the two enzymes. We have obtained high-resolution crystal structures of AKR4C8 (1.4 Å) and AKR4C9 (1.25 Å) in ternary complexes with NADP + and acetate. Three extended loops, present in all AKRs and responsible for defining the cofactor- and substrate-binding sites, are shorter in the 4C subfamily compared to other AKRs. Consequently, the crystal structures reveal open and accommodative substrate-binding sites, which correlates with their broad substrate specificity. It is suggested that the primary role of these enzymes may be to detoxify a range of toxic aldehydes and ketones produced during stress, although the precise nature of the principal natural substrates remains to be determined.

Arabidopsis thaliana wax synthase gene homologues show diverse expression patterns that suggest a specialized role for these genes in reproductive organs

Wax esters perform a wide variety of important functions in bacteria, plants, and animals. In plants, wax esters are integral components of the protective cuticles and also serve as primary carbon and energy storage compounds in the seeds of some species such as Simmondsia chinensis. Wax synthases catalyze wax ester formation from fatty acids and alcohols. While wax synthase activity has only been demonstrated for a few proteins, many putative wax synthases have been identified in sequence databases based on homology to known wax synthases. Twelve wax synthase homologues were previously identified in the Arabidopsis thaliana genome sequence. Establishing function(s) for these genes has proven difficult since prior studies failed to detect expression of any of these genes. Here we present results from reverse transcriptase PCR experiments and analysis of publicly available gene expression databases which show that eleven of these twelve homologues are expressed in Arabidopsis, with a bias toward expression in reproductive organs, and that one of the putative homologues is likely to be a pseudogene. These results are discussed relative to other previously described wax synthases as well as numerous new homologues that are being discovered as sequence databases expand.

Dual coding of siRNAs and miRNAs by plant transposable elements

We recently proposed a specific model whereby miRNAs encoded from short nonautonomous DNA-type TEs known as MITEs evolved from corresponding ancestral full-length (autonomous) elements that originally encoded short interfering (siRNAs). Our miRNA-origins model predicts that evolutionary intermediates may exist as TEs that encode both siRNAs and miRNAs, and we analyzed Arabidopsis thaliana and Oryza sativa (rice) genomic sequence and expression data to test this prediction. We found a number of examples of individual plant TE insertions that encode both siRNAs and miRNAs. We show evidence that these dual coding TEs can be expressed as readthrough transcripts from the intronic regions of spliced RNA messages. These TE transcripts can fold to form the hairpin (stem-loop) structures characteristic of miRNA genes along with longer double-stranded RNA regions that typically are processed as siRNAs. Taken together with a recent study showing Drosha independent processing of miRNAs from Drosophila introns, our results indicate that ancestral miRNAs could have evolved from TEs prior to the full elaboration of the miRNA biogenesis pathway. Later, as the specific miRNA biogenesis pathway evolved, and numerous other expressed inverted repeat regions came to be recognized by the miRNA processing endonucleases, the host gene-related regulatory functions of miRNAs emerged. In this way, host genomes were afforded an additional level of regulatory complexity as a by-product of TE defense mechanisms. The siRNA-to-miRNA evolutionary transition is representative of a number of other regulatory mechanisms that evolved to silence TEs and were later co-opted to serve as regulators of host gene expression.

Inventory, evolution and expression profiling diversity of the LEA (late embryogenesis abundant) protein gene family in Arabidopsis thaliana

We analyzed the Arabidopsis thaliana genome sequence to detect Late Embryogenesis Abundant (LEA) protein genes, using as reference sequences proteins related to LEAs previously described in cotton or which present similar characteristics. We selected 50 genes representing nine groups. Most of the encoded predicted proteins are small and contain repeated domains that are often specific to a unique LEA group. Comparison of these domains indicates that proteins with classical group 5 motifs are related to group 3 proteins and also gives information on the possible history of these repetitions. Chromosomal gene locations reveal that several LEA genes result from whole genome duplications (WGD) and that 14 are organized in direct tandem repeats. Expression of 45 of these genes was tested in different plant organs, as well as in response to ABA and in mutants (such as abi3, abi5, lec2 and fus3) altered in their response to ABA or in seed maturation. The results demonstrate that several so-called LEA genes are expressed in vegetative tissues in the absence of any abiotic stress, that LEA genes from the same group do not present identical expression profile and, finally, that regulation of LEA genes with apparently similar expression patterns does not systematically involve the same regulatory pathway.

Overexpression of Arabidopsis MAP kinase kinase 7 leads to activation of plant basal and systemic acquired resistance

There is a growing body of evidence indicating that mitogen-activated protein kinase (MAPK) cascades are involved in plant defense responses. Analysis of the completed Arabidopsis thaliana genome sequence has revealed the existence of 20 MAPKs, 10 MAPKKs and 60 MAPKKKs, implying a high level of complexity in MAPK signaling pathways, and making the assignment of gene functions difficult. The MAP kinase kinase 7 (MKK7) gene of Arabidopsis has previously been shown to negatively regulate polar auxin transport. Here we provide evidence that MKK7 positively regulates plant basal and systemic acquired resistance (SAR). The activation-tagged bud1 mutant, in which the expression of MKK7 is increased, accumulates elevated levels of salicylic acid (SA), exhibits constitutive pathogenesis-related (PR) gene expression, and displays enhanced resistance to both Pseudomonas syringae pv. maculicola (Psm) ES4326 and Hyaloperonospora parasitica Noco2. Both PR gene expression and disease resistance of the bud1 plants depend on SA, and partially depend on NPR1. We demonstrate that the constitutive defense response in bud1 plants is a result of the increased expression of MKK7, and requires the kinase activity of the MKK7 protein. We found that expression of the MKK7 gene in wild-type plants is induced by pathogen infection. Reducing mRNA levels of MKK7 by antisense RNA expression not only compromises basal resistance, but also blocks the induction of SAR. Intriguingly, ectopic expression of MKK7 in local tissues induces PR gene expression and resistance to Psm ES4326 in systemic tissues, indicating that activation of MKK7 is sufficient for generating the mobile signal of SAR.

The use of comparative genome analysis and syntenic relationships allows extrapolating the position of Zn tolerance QTL regions from Arabidopsis halleri into Arabidopsis thaliana

Arabidopsis halleri is a species that has undergone natural selection for zinc (Zn) tolerance. Isolation of the quantitative trait loci (QTL) associated with this trait holds great promise for the identification of the main genes responsible for this adaptation. Using a segregating progeny produced by an interspecific cross, we previously constructed a genetic linkage map of A. halleri × A. lyrata petraea and mapped the three main QTL that confer Zn tolerance in A. halleri (Willems et al.). The goal of the present study is to compare the genetic linkage map of A. halleri × A. l. petraea to the annotated A. thaliana genome sequence to generate a tool for A. halleri genomic approaches. To achieve this aim, we constructed a genetic linkage map with 81 markers anchored on A. thaliana, including 23 genes known to be involved in metal homeostasis. First, this provided an extensive overview of the chromosomal rearrangements that have occurred since the divergence between A. thaliana and its closest relative A. halleri. Second, on the basis of the syntenic relationships assessed experimentally through this work, we transferred the QTL confidence intervals for Zn tolerance to the A. thaliana physical map, allowing access to all the genes localized in the corresponding regions. Third, we validated from the 23 genes involved in metal homeostasis the three ones localized in the QTL regions that can be considered the best candidates for conferring Zn tolerance.

Comparison between a coffee single copy chromosomal region and Arabidopsis duplicated counterparts evidenced high level synteny between the coffee genome and the ancestral Arabidopsis genome

The Arabidopsis thaliana genome sequence provides a catalogue of reference genes that can be used for comparative analysis of other species thereby facilitating map-based cloning in economically important crops. We made use of a coffee bacterial artificial chromosome (BAC) contig linked to the S(H)3 leaf rust resistance gene to assess microsynteny between coffee (Coffea arabica L.) and Arabidopsis. Microsynteny was revealed and the matching counterparts to C. arabica contigs were seen to be scattered throughout four different syntenic segments of Arabidopsis on chromosomes (Ath) I, III, IV and V. Coffee BAC filter hybridizations were performed using coffee putative conserved orthologous sequences to Arabidopsis predicted genes located on the different Arabidopsis syntenic regions. The coffee BAC contig related to the S(H)3 region was successfully consolidated and later on validated by fingerprinting. Furthermore, the anchoring markers appeared in same order on the coffee BAC contigs and in all Arabidopsis segments with the exception of a single inversion on AtIII and AtIV Arabidopsis segments. However, the S(H)3 coffee region appears to be closer to the ancestral genome segment (before the divergence of Arabidopsis and coffee) than any of the duplicated counterparts in the present-day Arabidopsis genome. The genome duplication events at the origin of its Arabidopsis counterparts occurred most probably after the separation (i.e. 94 million years ago) of Euasterid (Coffee) and Eurosid (Arabidopsis).

Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 35

Catalysing the hydrolysis of terminal β-galactosyl residues from carbohydrates, galactolipids, and glycoproteins, glycoside hydrolase family 35 (β-galactosidases; BGALs) are widely distributed in plants and believed to play many key roles, including modification of cell wall components. Completion of the Arabidopsis thaliana genome sequencing project has, for the first time, allowed an examination of the total number, gene structure, and evolutionary patterns of all Family 35 members in a representative (model) angiosperm. Reiterative database searches established a multigene family of 17 members (designated BGAL1– BGAL17). Using these genes as query sequences, BLAST and Hidden Markov Model searches identified BGAL genes among 22 other eukaryotes, whose genomic sequences are known. The Arabidopsis ( n = 17) and rice ( n = 15) BGAL families were much larger than those of Chlamydomonas, fungi, and animals ( n = 0–4), and a lineage-specific expansion of BGAL genes apparently occurred after divergence of the Arabidopsis and rice lineages. All plant BGAL genes, with the exception of Arabidopsis BGAL17 and rice Os 9633.m04334, form a monophyletic group. Arabidopsis BGAL expression levels are much higher in mature leaves, roots, flowers, and siliques but are lower in young seedlings. BGAL8, BGAL11, BGAL13, BGAL14, and BGAL16 are expressed only in flowers. Catalytically active BGAL4 was produced in the E. coli and baculoviral expression systems, purified to electrophoretic homogeneity, and partially characterized. The purified enzyme hydrolyzed p- and o-nitrophenyl-β- d-galactosides. It also cleaved β-(1,3)-, β-(1,4)-, and β-(1,6)-linked galactobiosides and galactotriosides, showing a marked preference for β-(1,3)- and β-(1,4)-linkages.

A comparative linkage map of oilseed rape and its use for QTL analysis of seed oil and erucic acid content

We have developed a new DH mapping population for oilseed rape, named TNDH, using genetically and phenotypically diverse parental lines. We used the population in the construction of a high stringency genetic linkage map, consisting of 277 loci, for use in quantitative genetic analysis. A proportion of the markers had been used previously in the construction of linkage maps for Brassica species, thus permitting the alignment of maps. The map includes 68 newly developed Sequence Tagged Site (STS) markers targeted to the homologues of defined genes of A. thaliana. The use of these markers permits the alignment of our linkage map with the A. thaliana genome sequence. An additional 74 loci (31 newly developed STS markers and 43 loci defined by SSR and RFLP markers that had previously been used in published linkage maps) were added to the map. These markers increased the resolution of alignment of the newly constructed linkage map with existing Brassica linkage maps and the A. thaliana genome sequence. We conducted field trials with the TNDH population at two sites, and over 2 years, and identified reproducible QTL for seed oil content and erucic acid content. The results provide new insights into the genetic control of seed oil and erucic acid content in oilseed rape, and demonstrate the utility of the linkage map and population.

Heterologous Expression and Biochemical Characterization of a Polyamine Oxidase from Arabidopsis Involved in Polyamine Back Conversion

Polyamine oxidase (PAO) is a flavin adenine dinucleotide-dependent enzyme involved in polyamine catabolism. Animal PAOs oxidize spermine (Spm), spermidine (Spd), and/or their acetyl derivatives to produce H2O2, an aminoaldehyde, and Spd or putrescine, respectively, thus being involved in a polyamine back-conversion pathway. On the contrary, plant PAOs that have been characterized to date oxidize Spm and Spd to produce 1,3-diaminopropane, H2O2, and an aminoaldehyde and are therefore involved in the terminal catabolism of polyamines. A database search within the Arabidopsis (Arabidopsis thaliana) genome sequence showed the presence of a gene (AtPAO1) encoding for a putative PAO with 45% amino acid sequence identity with maize (Zea mays) PAO. The AtPAO1 cDNA was isolated and cloned in a vector for heterologous expression in Escherichia coli. The recombinant protein was purified by affinity chromatography on guazatine-Sepharose 4B and was shown to be a flavoprotein able to oxidize Spm, norspermine, and N1-acetylspermine with a pH optimum at 8.0. Analysis of the reaction products showed that AtPAO1 produces Spd from Spm and norspermidine from norspermine, demonstrating a substrate oxidation mode similar to that of animal PAOs. To our knowledge, AtPAO1 is the first plant PAO reported to be involved in a polyamine back-conversion pathway.

Gain-of-Function Phenotypes of Many CLAVATA3/ESR Genes, Including Four New Family Members, Correlate with Tandem Variations in the Conserved CLAVATA3/ESR Domain

Secreted peptide ligands are known to play key roles in the regulation of plant growth, development, and environmental responses. However, phenotypes for surprisingly few such genes have been identified via loss-of-function mutant screens. To begin to understand the processes regulated by the CLAVATA3 (CLV3)/ESR (CLE) ligand gene family, we took a systems approach to gene identification and gain-of-function phenotype screens in transgenic plants. We identified four new CLE family members in the Arabidopsis (Arabidopsis thaliana) genome sequence and determined their relative transcript levels in various organs. Overexpression of CLV3 and the 17 CLE genes we tested resulted in premature mortality and/or developmental timing delays in transgenic Arabidopsis plants. Overexpression of 10 CLE genes and the CLV3 positive control resulted in arrest of growth from the shoot apical meristem (SAM). Overexpression of nearly all the CLE genes and CLV3 resulted in either inhibition or stimulation of root growth. CLE4 expression reversed the SAM proliferation phenotype of a clv3 mutant to one of SAM arrest. Dwarf plants resulted from overexpression of five CLE genes. Overexpression of new family members CLE42 and CLE44 resulted in distinctive shrub-like dwarf plants lacking apical dominance. Our results indicate the capacity for functional redundancy of many of the CLE ligands. Additionally, overexpression phenotypes of various CLE family members suggest roles in organ size regulation, apical dominance, and root growth. Similarities among overexpression phenotypes of many CLE genes correlate with similarities in their CLE domain sequences, suggesting that the CLE domain is responsible for interaction with cognate receptors.

Plant molecular biology in China: Opportunities and challenges

In the 21st century, mankind has witnessed great advances in life sciences, including completion of theArabidopsis thaliana genome sequence and major advances with the rice genome. But, along with global economic development, urbanization, and depletion of natural resources, many serious problems are emerging (for example, environment, food, population, energy), which reinforce the need for sustainable development in many countries and in many institutions and prompt progress in life sciences globally. Plants offer the globe its only renewable resource of food, building material, and energy. Plants have highly sophisticated, concerted, short- and long-term adaptive mechanisms to the environment. Plants have great importance in global sustainable economic development. Plant molecular biology is a most essential and powerful tool in this process. Globally, plant molecular biology research is progressing rapidly, from use of model plants to cereal crops and from cellular processes to evolutionary mechanisms. Results of these studies have value in ecosystem regulation and environmental phytoremediation. China is a large agricultural country with one-fifth of the world's population. The gap for the level of plant molecular biology research in China is large, compared with that in other developed nations. However, some Chinese laboratories (notably those of academicians Jiayang Li, Zhihong Xu, Zhensheng Li, Mengmin Hong, Qifa Zhang and professors Yonbiao Xue, Shouyi Chen, and Zhen Zhu) have kept pace with international developments in plant molecular biology. How to fully utilize plant biodiversity in China requires future advances in plant molecular biology. This minireview discusses opportunities and challenges in plant molecular biology in China, analyzes the current status of international plant molecular biology, and provides suggestions to accelerate and advance international efforts.

Evolutionary relationship and substrate specificity of Arabidopsis thaliana fatty acid omega-hydroxylase

On the basis of a phylogenic tree constructed using the available Arabidopsis thaliana genomic sequence, new fatty acid (FA) omega-hydroxylases were cloned and expressed in yeast. Several uncharacterized cytochrome P450s (CYP, P450), comprising a phylogenic sub-cluster (CYP86A, 94B, 94C, 96A, 704A, 97B and 711A) demonstrated FA omega-hydroxylase activities towards saturated FAs with medium chain length. While CYP96A4 showed the highest catalytic activity among the enzymes characterized in this study, other CYP96A subfamily members did not display any potent activity. In addition, CYP704A2 and CYP711A1, which are phylogenetically distant to both CYP86 and CYP94 with FA omega-hydroxylase activities, did not show any metabolic conversion of FAs. As we have studied omega-hydroxylation of FAs catalyzed by a series of CYP94 isoforms, active site models were produced to compare inactive CYP704A2 and CYP711A1 to active CYP94C1. The modeled structures revealed that the hydrophobicity in the heme binding site is very different between active and inactive isoforms. CYP94C1 contains highly hydrophobic residues while CYP704A2 and CYP711A1 do not. Our study provides evidence that substrate specificity is conserved in phylogenetically related isoforms and suggests specific residues in the active site pocket that play a key role in determining substrate specificity.

Comparing low coverage random shotgun sequence data fromBrassica oleraceaandOryza sativagenome sequence for their ability to add to the annotation ofArabidopsis thaliana

Since the completion of the Arabidopsis thaliana genome sequence, there is an ongoing effort to annotate the genome as accurately as possible. Comparing genome sequences of related species complements the current annotation strategies by identifying genes and improving gene structure. A total of 595,321 Brassica oleracea shotgun reads were sequenced by TIGR (The Institute for Genome Research) and the collaboration of Washington University and Cold Spring Harbor. Vicogenta (a genome viewer based on GMOD and GBrowse) was created to view the current annotation and sequence alignments for Arabidopsis. Brassica reads were compared with the Arabidopsis genome and proteome databases using BLAST. Hypothetical genes and conserved unannotated regions on the short arm of chromosome 4 from Arabidopsis were experimentally verified using RT-PCR. We were able to improve the Arabidopsis annotation by identifying 25 genes that were missed, and confirming expression of 43 hypothetical genes in Arabidopsis. We were also able to detect conservation in genes whose transcription is normally suppressed due to methylation. We also examined how useful the O. sativa genome and ESTs from other species are, compared with Brassica, in improving the Arabidopsis annotation.