David Patton

A High-Throughput Arabidopsis Reverse Genetics System

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from ∼100,000 transformed lines. A total of 85,108 TAIL-PCR products from 52,964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.

Identification of Genes Required for Embryo Development in Arabidopsis

A long-term goal of Arabidopsis research is to define the minimal gene set needed to produce a viable plant with a normal phenotype under diverse conditions. This will require both forward and reverse genetics along with novel strategies to characterize multigene families and redundant biochemical pathways. Here we describe an initial dataset of 250 EMB genes required for normal embryo development in Arabidopsis. This represents the first large-scale dataset of essential genes in a flowering plant. When compared with 550 genes with other knockout phenotypes, EMB genes are enriched for basal cellular functions, deficient in transcription factors and signaling components, have fewer paralogs, and are more likely to have counterparts among essential genes of yeast (Saccharomyces cerevisiae) and worm (Caenorhabditis elegans). EMB genes also represent a valuable source of plant-specific proteins with unknown functions required for growth and development. Analyzing such unknowns is a central objective of genomics efforts worldwide. We focus here on 34 confirmed EMB genes with unknown functions, demonstrate that expression of these genes is not embryo-specific, validate a strategy for identifying interacting proteins through complementation with epitope-tagged proteins, and discuss the value of EMB genes in identifying novel proteins associated with important plant processes. Based on sequence comparison with essential genes in other model eukaryotes, we identify 244 candidate EMB genes without paralogs that represent promising targets for reverse genetics. These candidates should facilitate the recovery of additional genes required for seed development.

FPA, a Gene Involved in Floral Induction in Arabidopsis, Encodes a Protein Containing RNA-Recognition Motifs

FPA is a gene that regulates flowering time in Arabidopsis via a pathway that is independent of daylength (the autonomous pathway). Mutations in FPA result in extremely delayed flowering. FPA was identified by means of positional cloning. The predicted FPA protein contains three RNA recognition motifs in the N-terminal region. FPA is expressed most strongly in developing tissues, similar to the expression of FCA and LUMINIDEPENDENS, two components of the autonomous pathway previously identified. Overexpression of FPA in Arabidopsis causes early flowering in noninductive short days and creates plants that exhibit a more day-neutral flowering behavior.

Embryonic Lethals and T-DNA Insertional Mutagenesis in Arabidopsis.

T-DNA insertional mutagenesis represents a promising approach to the molecular isolation of genes with essential functions during plant embryo development. We describe in this report the isolation and characterization of 18 mutants of Arabidopsis thaliana defective in embryo development following seed transformation with Agrobacterium tumefaciens. Random T-DNA insertion was expected to result in a high frequency of recessive embryonic lethals because many target genes are required for embryogenesis. The cointegrate Ti plasmid used in these experiments contained the nopaline synthase and neomycin phosphotransferase gene markers. Nopaline assays and resistance to kanamycin were used to estimate the number of functional inserts present in segregating families. Nine families appeared to contain a T-DNA insert either within or adjacent to the mutant gene. Eight families were clearly not tagged with a functional insert and appeared instead to contain mutations induced during the transformation process. DNA gel blot hybridization with internal and right border probes revealed a variety of rearrangements associated with T-DNA insertion. A general strategy is presented to simplify the identification of tagged embryonic mutants and facilitate the molecular isolation of genes required for plant embryogenesis.

The Arabidopsis SeedGenes Project

The SeedGenes database (http://www.seedgenes.org) presents molecular and phenotypic information on essential, non-redundant genes of Arabidopsis that give a seed phenotype when disrupted by mutation. Experimental details are synthesized for efficient use by the community and organized into two major sections in the database, one dealing with genes and the other with mutant alleles. The database can be queried for detailed information on a single gene to create a SeedGenes Profile. Queries can also generate lists of genes or mutants that fit specified criteria. The long-term goal is to establish a complete collection of Arabidopsis genes that give a knockout phenotype. This information is needed to focus attention on genes with important cellular functions in a model plant and to assess from a genetic perspective the extent of functional redundancy in the Arabidopsis genome.

The TITAN5 Gene of Arabidopsis Encodes a Protein Related to the ADP Ribosylation Factor Family of GTP Binding Proteins

The titan (ttn) mutants of Arabidopsis exhibit dramatic alterations in mitosis and cell cycle control during seed development. Endosperm development in these mutants is characterized by the formation of giant polyploid nuclei with enlarged nucleoli. Embryo development is accompanied by significant cell enlargement in some mutants (ttn1 and ttn5) but not others (ttn2 and ttn3). We describe here the molecular cloning of TTN5 using a T-DNA–tagged allele. A second allele with a similar phenotype contains a nonsense mutation in the same coding region. The predicted protein is related to ADP ribosylation factors (ARFs), members of the RAS family of small GTP binding proteins that regulate various cellular functions in eukaryotes. TTN5 is most closely related in sequence to the ARL2 class of ARF-like proteins isolated from humans, rats, and mice. Although the cellular functions of ARL proteins remain unclear, the ttn5 phenotype is consistent with the known roles of ARFs in the regulation of intracellular vesicle transport.

Mapping genes essential for embryo development in Arabidopsis thaliana

SummaryWe have previously isolated and characterized over 90 recessive mutants of Arabidopsis thaliana defective in embryo development. These emb mutants have been shown to differ in lethal phase, extent of abnormal development, and response in culture. We demonstrate in this report the value and efficiency of mapping emb genes relative to visible and molecular markers. Sixteen genes essential for embryo development were mapped relative to visible markers by analyzing progeny of selfed F1 plants. Embryonic lethals are now the most common type of visible marker included on the linkage map of Arabidopsis. Backcrosses were used in several cases to orient genes relative to adjacent markers. Three genes were located to chromosome arms with telotrisomics by screening for a reduction in the percentage of aborted seeds produced by F1 plants. A restriction fragment length polymorphism (RFLP) mapping strategy that utilizes pooled EMB/EMB F2 plants was devised to increase the efficiency of mapping embryonic lethals relative to molecular markers. This strategy was tested by demonstrating that the biol locus of Arabidopsis is within 0.5 cM of an existing RFLP marker. Mapping embryonic lethals with both visible and molecular markers may therefore help to identify large numbers of genes with essential functions in Arabidopsis.

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 biochemical stimuli.

High-frequency plant regeneration from cultured cotyledons of Arabidopsis thaliana

Wild-type plants of Arabidopsis thaliana strain “Columbia” regenerated at a high frequency from immature cotyledons cultured on a shoot-inducing medium containing 1.0 mg/l 6-benzylaminopurine and 0.1 mg/l 1-naphthaleneacetic acid. Cotyledon segments expanded rapidly and produced numerous shoots after 2–3 weeks in culture. Regeneration occurred in the absence of the original shoot apex. Hypocotyl segments from immature embryos produced root hairs and callus in culture but only rarely developed shoots. Hygromycin, kanamycin and G-418 inhibited cotyledon expansion and shoot formation in culture. Vancomycin was much less toxic to cotyledon segments than either carbenicillin or cefotaxime. Immature cotyledons therefore yield numerous regenerated plants that may be useful in future transformation studies.

In vitro morphogenesis of arrested embryos from lethal mutants of Arabidopsis thaliana.

SummaryArrested embryos from lethal (emb) mutants of Arabidopsis thaliana were rescued on a nutrient medium designed to promote plant regeneration from immature wild-type cotyledons. The best response was observed with mutant embryos arrested at the heart to cotyledon stages of development. Embryos arrested at a globular stage produced callus but failed to turn green or form normal shoots in culture. Many of the mutant plants produced in culture were unusually pale with abnormal leaves, rosettes, and patterns of reproductive development. Other plants were phenotypically normal except for the presence of siliques containing 100% aborted seeds following self-pollination. These results demonstrate that genes with essential functions during plant embryo development differ in their pattern of expression at later stages of the life cycle. Most of the 15 genes examined in this study were essential for embryogenesis but were required again for subsequent stages of development. Only EMB24 appeared to be limited in function to embryo development. These differences in the response of mutant embryos in culture may facilitate the classification of embryonic lethals and the identification of genes with developmental rather than housekeeping functions.