FOX-superroots of Lotus corniculatus, overexpressing Arabidopsis full-length cDNA, show stable variations in morphological traits

Using the full-length cDNA overexpressor (FOX) gene-hunting system, we have generated 130 Arabidopsis FOX-superroot lines in bird's-foot trefoil (Lotus corniculatus) for the systematic functional analysis of genes expressed in roots and for the selection of induced mutants with interesting root growth characteristics. We used the Arabidopsis-FOX Agrobacterium library (constructed by ligating pBIG2113SF) for the Agrobacterium-mediated transformation of superroots (SR) and the subsequent selection of gain-of-function mutants with ectopically expressed Arabidopsis genes. The original superroot culture of L. corniculatus is a unique host system displaying fast root growth in vitro, allowing continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions. Several of the Arabidopsis FOX-superroot lines show interesting deviations from normal growth and morphology of roots from SR-plants, such as differences in pigmentation, growth rate, length or diameter. Some of these mutations are of potential agricultural interest. Genomic PCR analysis revealed that 100 (76.9%) out of the 130 transgenic lines showed the amplification of single fragments. Sequence analysis of the PCR fragments from these 100 lines identified full-length cDNA in 74 of them. Forty-three out of 74 full-length cDNA carried known genes. The Arabidopsis FOX-superroot lines of L. corniculatus, produced in this study, expand the FOX hunting system and provide a new tool for the genetic analysis and control of root growth in a leguminous forage plant.     

Systematic approaches to using the FOX hunting system to identify useful rice genes

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23 000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis . This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.     

RiceFOX: a database of Arabidopsis mutant lines overexpressing rice full-length cDNA that contains a wide range of trait information to facilitate analysis of gene function

Identification of gene function is important not only for basic research but also for applied science, especially with regard to improvements in crop production. For rapid and efficient elucidation of useful traits, we developed a system named FOX hunting (Full-length cDNA Over-eXpressor gene hunting) using full-length cDNAs (fl-cDNAs). A heterologous expression approach provides a solution for the high-throughput characterization of gene functions in agricultural plant species. Since fl-cDNAs contain all the information of functional mRNAs and proteins, we introduced rice fl-cDNAs into Arabidopsis plants for systematic gain-of-function mutation. We generated >30,000 independent Arabidopsis transgenic lines expressing rice fl-cDNAs (rice FOX Arabidopsis mutant lines). These rice FOX Arabidopsis lines were screened systematically for various criteria such as morphology, photosynthesis, UV resistance, element composition, plant hormone profile, metabolite profile/fingerprinting, bacterial resistance, and heat and salt tolerance. The information obtained from these screenings was compiled into a database named 'RiceFOX'. This database contains around 18,000 records of rice FOX Arabidopsis lines and allows users to search against all the observed results, ranging from morphological to invisible traits. The number of searchable items is approximately 100; moreover, the rice FOX Arabidopsis lines can be searched by rice and Arabidopsis gene/protein identifiers, sequence similarity to the introduced rice fl-cDNA and traits. The RiceFOX database is available at http://ricefox.psc.riken.jp/.     

Generation of Arabidopsis Mutants by Heterologous Expression of a Full-Length cDNA Library from Tomato Fruits

Heterologous expression of cDNA library in Arabidopsis and other plants has been used for gene identifications. To identify functions of tomato genes, we expressed a tomato full-length cDNA library in Arabidopsis thaliana and generated over 7,000 mutants. We constructed a tomato cDNA library with a plant transformation-ready binary vector that contained a higher percentage of full-length cDNAs since synthesized double-stranded cDNA was size-selected using gel electrophoresis, with cDNA sizes of 2–5 kb being gel-purified for ligation onto the binary vector. Sequencing of 81 cDNA clones indicates that 75% (61) are full-length genes, which is similar to sequencing of inserted cDNA in Arabidopsis. The library was used to transform Arabidopsis plants. Among the 7,000 mutants, one was found to be a dwarf due to the expression of an ATP synthase, and another vegetative mutant did not produce flowers even after 7 months. The technique was validated by reintroducing the tomato ribosomal protein L9 gene and can be used in any other plant species as a gene discovery tool.     

A genome-wide gain-of-function analysis of rice genes using the FOX-hunting system

The latest report has estimated the number of rice genes to be approximately 32,000. To elucidate the functions of a large population of rice genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the Full-length cDNA Over-eXpresser (FOX) gene-hunting system. This system is very useful for analyzing various gain-of-function phenotypes from large populations of transgenic plants overexpressing cDNAs of interest and others with unknown or important functions. We collected the plasmid DNAs of 13,980 independent full-length cDNA (FL-cDNA) clones to produce a FOX library by placing individual cDNAs under the control of the maize Ubiquitin-1 promoter. The FOX library was transformed into rice by Agrobacterium-mediated high-speed transformation. So far, we have generated approximately 12,000 FOX-rice lines. Genomic PCR analysis indicated that the average number of FL-cDNAs introduced into individual lines was 1.04. Sequencing analysis of the PCR fragments carrying FL-cDNAs from 8615 FOX-rice lines identified FL-cDNAs in 8225 lines, and a database search classified the cDNAs into 5462 independent ones. Approximately 16.6% of FOX-rice lines examined showed altered growth or morphological characteristics. Three super-dwarf mutants overexpressed a novel gibberellin 2-oxidase gene,confirming the importance of this system. We also show here the other morphological alterations caused by individual FL-cDNA expression. These dominant phenotypes should be valuable indicators for gene discovery and functional analysis.     

Isolating the Arabidopsis thaliana genes for de novo purine synthesis by suppression of Escherichia coli mutants. I. 5'-Phosphoribosyl-5-aminoimidazole synthetase.

We have initiated an investigation of the de novo purine nucleotide biosynthetic pathway in the plant Arabidopsis thaliana. Functional suppression of Escherichia coli auxotrophs allowed the direct isolation of expressed Arabidopsis leaf cDNAs. Using this approach we have successfully suppressed mutants in 4 of the 12 genes in this pathway. One of these cDNA clones, encoding 5'-phosphoribosyl-5-aminoimidazole (AIR) synthetase (PUR5) has been characterized in detail. Analysis of genomic DNA suggests that the Arabidopsis genome contains a single AIR synthetase gene. Analysis of the cDNA sequence and mRNA size suggests that this enzyme activity is encoded by a monofunctional polypeptide, similar to that of bacteria and unlike other eukaryotes. The Arabidopsis AIR synthetase contains a basic hydrophobic transit peptide consistent with transport into chloroplasts. Comparison of both the predicted amino acid and nucleotide sequence from Arabidopsis to those of eight other distant organisms suggests that the plant sequence is more similar to the bacterial sequences than to other eukaryotic sequences. This study provides the groundwork for future investigations into the regulation of de novo purine biosynthesis in plants. Additionally, we have demonstrated that functional suppression of bacterial mutants may provide a useful method for cloning a variety of plant genes.     

Chemical regulated production of cDNAs from genomic DNA fragments in plants

We have developed a new chemical inducible genetic system that allows for the isolation of any cDNA molecule from in vitro generated genomic transgenes in transgenic plants. This system, termed regulated in vivo cDNA generation (RIDE), permits both targeted isolation of individual full-length cDNA molecules and random isolation of any partial or full-length cDNA from in planta genomic libraries. The RIDE system makes use of the 17-beta estradiol-inducible promoter system linked to intron donor and acceptor sites in a new binary vector configuration. In transgenic Arabidopsis and tobacco plants, we show that the RIDE system can isolate low-abundance full-length cDNAs previously unattainable by conventional means at high efficiencies (75-85%). The ability to randomly isolate individual exons and exons spliced together from genomic libraries in planta suggest that this system can be used for the isolation of any cDNA molecules. The RIDE system thus appears to be an efficient and versatile system for the generation of potentially any cDNA molecule. Moreover, the ORF structural data generated will be of value in both verifying and correcting computational ORF predications in the databases available to the scientific community.     

Construction of a full-length cDNA library from young spikelets of hexaploid wheat and its characterization by large-scale sequencing of expressed sequence tags

The polyploid nature of wheat is a key characteristic of the plant. Full-length complementary DNAs (cDNAs) provide essential information that can be used to annotate the genes and provide a functional analysis of these genes and their products. We constructed a full-length cDNA library derived from young spikelets of common wheat, and obtained 24056 expressed sequence tags (ESTs) from both ends of the cDNA clones. These ESTs were grouped into 3605 contigs using the phrap method, representing expressed loci from each of the three genomes. Using BLAST, 3605 contigs were grouped into 1902 gene clusters, showing that loci of the three genomes are not always expressed. A homology search of these gene clusters against a wheat EST database (15964 gene clusters) and a rice full-length cDNA database (21447 gene clusters) revealed that a quarter of the wheat full-length cDNAs were novel. A protein database of Arabidopsis was used to examine the functional classification of these gene clusters. The GC-content in the 5 -UTR region of wheat cDNAs was compared to that of rice. Forty-three genes (3.5% of wheat cDNAs homologous to those of rice) possessed distinct GC-content in the 5 -UTR region, suggesting different breeding behaviors of wheat and rice.     

Expression, purification, and characterization of the protein repair l-isoaspartyl methyltransferase from Arabidopsis thaliana

Protein l-isoaspartate (d-aspartate) O-methyltransferase (EC 2.1.1. 77) is a repair enzyme that methylates abnormal l-isoaspartate residues in proteins which arise spontaneously as a result of aging. This enzyme initiates their conversion back into the normal l-aspartate form by a methyl esterification reaction. Previously, partial cDNAs of this enzyme were isolated from the higher plant Arabidopsis thaliana. In this study, we report the cloning and expression of a full-length cDNA of l-isoaspartyl methyltransferase from A. thaliana into Escherichia coli under the P(BAD) promoter, which offers a high level of expression under a tight regulatory control. The enzyme is found largely in the soluble fraction. We purified this recombinant enzyme to homogeneity using a series of steps involving DEAE-cellulose, gel filtration, and hydrophobic interaction chromatographies. The homogeneous enzyme was found to have maximum activity at 45 degrees C and a pH optimum from 7 to 8. The enzyme was found to have a wide range of affinities for l-isoaspartate-containing peptides and displayed relatively poor reactivity toward protein substrates. The best methyl-accepting substrates were KASA-l-isoAsp-LAKY (K(m) = 80 microM) and VYP-l-isoAsp-HA (K(m) = 310 microM). We also expressed the full-length form and a truncated version of this enzyme (lacking the N-terminal 26 amino acid residues) in E. coli under the T7 promoter. Both the full-length and the truncated forms were active, though overexpression of the truncated enzyme led to a complete loss of activity.     

Construction of a human full-length cDNA bank

We aimed to construct a full-length cDNA bank from an entire set of human genes and to analyze the function of a protein encoded by each cDNA. To achieve this purpose, a multifunctional phagemid shuttle vector, pKAl, was constructed for preparing a high-quality cDNA library composed of full-length cDNA clones which can be sequenced and expressed in vitro and in mammalian cells without subcloning the cDNA fragment into other vectors. Using this as a vector primer, we have prepared a prototype of the bank composed of full-length cDNAs encoding 236 human proteins whose amino acid sequences are identical or similar to known proteins. Most cDNAs contain a putative cap site sequence, some of which show a pyrimidine-rich conserved sequence exhibiting polymorphism. It was confirmed that the vector permits efficient in vitro translation, expression in mammalian cells and the preparation of nested deletion mutants.     

A first survey of the rye (Secale cereale) genome composition through BAC end sequencing of the short arm of chromosome 1R

Background

Thellungiella halophila (also known as Thellungiella salsuginea) is a model halophyte with a small plant size, short life cycle, and small genome. It easily undergoes genetic transformation by the floral dipping method used with its close relative, Arabidopsis thaliana. Thellungiella genes exhibit high sequence identity (approximately 90% at the cDNA level) with Arabidopsis genes. Furthermore, Thellungiella not only shows tolerance to extreme salinity stress, but also to chilling, freezing, and ozone stress, supporting the use of Thellungiella as a good genomic resource in studies of abiotic stress tolerance.

Results

We constructed a full-length enriched Thellungiella (Shan Dong ecotype) cDNA library from various tissues and whole plants subjected to environmental stresses, including high salinity, chilling, freezing, and abscisic acid treatment. We randomly selected about 20 000 clones and sequenced them from both ends to obtain a total of 35 171 sequences. CAP3 software was used to assemble the sequences and cluster them into 9569 nonredundant cDNA groups. We named these cDNAs "RTFL" (RIKEN Thellungiella Full-Length) cDNAs. Information on functional domains and Gene Ontology (GO) terms for the RTFL cDNAs were obtained using InterPro. The 8289 genes assigned to InterPro IDs were classified according to the GO terms using Plant GO Slim. Categorical comparison between the whole Arabidopsis genome and Thellungiella genes showing low identity to Arabidopsis genes revealed that the population of Thellungiella transport genes is approximately 1.5 times the size of the corresponding Arabidopsis genes. This suggests that these genes regulate a unique ion transportation system in Thellungiella.

Conclusion

As the number of Thellungiella halophila (Thellungiella salsuginea) expressed sequence tags (ESTs) was 9388 in July 2008, the number of ESTs has increased to approximately four times the original value as a result of this effort. Our sequences will thus contribute to correct future annotation of the Thellungiella genome sequence. The full-length enriched cDNA clones will enable the construction of overexpressing mutant plants by introduction of the cDNAs driven by a constitutive promoter, the complementation of Thellungiella mutants, and the determination of promoter regions in the Thellungiella genome.     

Efficient and high-throughput vector construction and Agrobacterium-mediated transformation of Arabidopsis thaliana suspension-cultured cells for functional genomics

We established a large-scale, high-throughput protocol to construct Arabidopsis thaliana suspension-cultured cell lines, each of which carries a single transgene, using Agrobacterium-mediated transformation. We took advantage of RIKEN Arabidopsis full-length (RAFL) cDNA clones and the Gateway cloning system for high-throughput preparation of binary vectors carrying individual full-length cDNA sequences. Throughout all cloning steps, multiple-well plates were used to treat 96 samples simultaneously in a high-throughput manner. The optimal conditions for Agrobacterium-mediated transformation of 96 independent binary vector constructs were established to obtain transgenic cell lines efficiently. We evaluated the protocol by generating transgenic Arabidopsis T87 cell lines carrying individual 96 metabolism-related RAFL cDNA fragments, and showed that the protocol was useful for high-throughput and large-scale production of gain-of-function lines for functional genomics.