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.

Arabidopsis cyt1 mutants are deficient in a mannose-1-phosphate guanylyltransferase and point to a requirement of N-linked glycosylation for cellulose biosynthesis.

Arabidopsis cyt1 mutants have a complex phenotype indicative of a severe defect in cell wall biogenesis. Mutant embryos arrest as wide, heart-shaped structures characterized by ectopic accumulation of callose and the occurrence of incomplete cell walls. Texture and thickness of the cell walls are irregular, and unesterified pectins show an abnormally diffuse distribution. To determine the molecular basis of these defects, we have cloned the CYT1 gene by a map-based approach and found that it encodes mannose-1-phosphate guanylyltransferase. A weak mutation in the same gene, called vtc1, has previously been identified on the basis of ozone sensitivity due to reduced levels of ascorbic acid. Mutant cyt1 embryos are deficient in N-glycosylation and have an altered composition of cell wall polysaccharides. Most notably, they show a 5-fold decrease in cellulose content. Characteristic aspects of the cyt1 phenotype, including radial swelling and accumulation of callose, can be mimicked with the inhibitor of N-glycosylation, tunicamycin. Our results suggest that N-glycosylation is required for cellulose biosynthesis and that a deficiency in this process can account for most phenotypic features of cyt1 embryos.

The Arabidopsis Information Resource (TAIR): a comprehensive database and web-based information retrieval, analysis, and visualization system for a model plant.

Arabidopsis thaliana, a small annual plant belonging to the mustard family, is the subject of study by an estimated 7000 researchers around the world. In addition to the large body of genetic, physiological and biochemical data gathered for this plant, it will be the first higher plant genome to be completely sequenced, with completion expected at the end of the year 2000. The sequencing effort has been coordinated by an international collaboration, the Arabidopsis Genome Initiative (AGI). The rationale for intensive investigation of Arabidopsis is that it is an excellent model for higher plants. In order to maximize use of the knowledge gained about this plant, there is a need for a comprehensive database and information retrieval and analysis system that will provide user-friendly access to Arabidopsis information. This paper describes the initial steps we have taken toward realizing these goals in a project called The Arabidopsis Information Resource (TAIR) (www.arabidopsis.org).

Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana.

The purpose of this project was to identify large numbers of Arabidopsis genes with essential functions during seed development. More than 120,000 T-DNA insertion lines were generated following Agrobacterium-mediated transformation. Transgenic plants were screened for defective seeds and putative mutants were subjected to detailed analysis in subsequent generations. Plasmid rescue and TAIL-PCR were used to recover plant sequences flanking insertion sites in tagged mutants. More than 4200 mutants with a wide range of seed phenotypes were identified. Over 1700 of these mutants were analyzed in detail. The 350 tagged embryo-defective (emb) mutants identified to date represent a significant advance toward saturation mutagenesis of EMB genes in Arabidopsis. Plant sequences adjacent to T-DNA borders in mutants with confirmed insertion sites were used to map genome locations and establish tentative identities for 167 EMB genes with diverse biological functions. The frequency of duplicate mutant alleles recovered is consistent with a relatively small number of essential (EMB) genes with nonredundant functions during seed development. Other functions critical to seed development in Arabidopsis may be protected from deleterious mutations by extensive genome duplications.

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.

The titan mutants of Arabidopsis are disrupted in mitosis and cell cycle control during seed development.

We describe in this report a novel class of mutants that should facilitate the identification of genes required for progression through the mitotic cell cycle during seed development in angiosperms. Three non-allelic titan (ttn) mutants with related but distinct phenotypes are characterized. The common feature among these mutants is that endosperm nuclei become greatly enlarged and highly polyploid. The mutant embryo is composed of a few giant cells in ttn1, several small cells in ttn2, and produces a normal plant in ttn3. Condensed chromosomes arrested at prophase of mitosis are found in the free nuclear endosperm of ttn1 and ttn2 seeds. Large mitotic figures with excessive numbers of chromosomes are visible in ttn3 endosperm. The ttn1 mutation appears to disrupt cytoskeletal organization because endosperm nuclei fail to migrate to the chalazal end of the seed. How double fertilization leads to the establishment of distinct patterns of mitosis and cytokinesis in the embryo and endosperm is a central question in plant reproductive biology. Molecular isolation of TITAN genes should help to answer this question, as well as related issues concerning cell cycle regulation, chromosome movement and endosperm identity in angiosperms.

Arabidopsis thaliana: a model plant for genome analysis.

Arabidopsis thaliana is a small plant in the mustard family that has become the model system of choice for research in plant biology. Significant advances in understanding plant growth and development have been made by focusing on the molecular genetics of this simple angiosperm. The 120-megabase genome of Arabidopsis is organized into five chromosomes and contains an estimated 20,000 genes. More than 30 megabases of annotated genomic sequence has already been deposited in GenBank by a consortium of laboratories in Europe, Japan, and the United States. The entire genome is scheduled to be sequenced by the end of the year 2000. Reaching this milestone should enhance the value of Arabidopsis as a model for plant biology and the analysis of complex organisms in general.

A cytokinesis-defective mutant of Arabidopsis (cyt1) characterized by embryonic lethality, incomplete cell walls, and excessive callose accumulation.

The genetic control of cell division in eukaryotes has been addressed in part through the analysis of cytokinesis-defective mutants. Two allelic mutants of Arabidopsis (cyt1-1 and cyt1-2) altered in cytokinesis and cell-wall architecture during embryogenesis are described in this report. Mutant embryos appear slightly abnormal at the heart stage and then expand to form a somewhat disorganized mass of enlarged cells with occasional incomplete walls. In contrast to the keule and knolle mutants of Arabidopsis and the cyd mutant of pea, which also exhibit defects in cytokinesis during embryogenesis, cyt1 embryos cannot be rescued in culture, are desiccation-intolerant at maturity, and produce cell walls with excessive callose as revealed through staining with the aniline blue fluorochrome, Sirofluor. Some cyt1 defects can be partially phenocopied by treatment with the herbicide dichlobenil, which is thought to interfere with cellulose biosynthesis. The distribution of unesterified pectins in cyt1 cell walls is also disrupted as revealed through immunocytochemical localization of JIM 5 antibodies. These features indicate that CYT1 plays an essential and unique role in plant growth and development and the establishment of normal cell-wall architecture.

Embryogenic transformation of the suspensor in twin, a polyembryonic mutant of Arabidopsis.

Spontaneous twinning is a widespread but infrequent phenomenon in higher plants. We describe here a mutant of Arabidopsis thaliana, twin, that yields an unusually high frequency of viable twin and occasional triplet seedlings. Supernumerary embryos of twin arise through a novel mechanism: transformation of cells within the suspensor, a differentiated structure established early in embryogenesis. Twin embryos develop in tandem within the seed, connected by intact segments of the suspensor. Transformed suspensor cells appear to duplicate the patterns of cell division and developmental pathways characteristic of zygotic embryogenesis. In addition to polyembryony, mutant embryos exhibit a number of developmental defects, including irregular patterns of cell division and abnormal morphology. The TWIN locus therefore appears to be required for normal development of the embryo proper as well as suppression of embryogenic potential in the suspensor. The development of viable secondary embryos in twin demonstrates that cells of the Arabidopsis suspensor can successfully establish embryonic polarity and complete the full spectrum of developmental programs normally restricted to the embryo proper. In addition, the twin phenotype indicates that disruption of a single genetic locus can result in the conversion of a single terminally differentiated cell type to an embryogenic state.

Leafy Cotyledon Mutants of Arabidopsis.

We have previously described a homeotic leafy cotyledon (lec) mutant of Arabidopsis that exhibits striking defects in embryonic maturation and produces viviparous embryos with cotyledons that are partially transformed into leaves. In this study, we present further details on the developmental anatomy of mutant embryos, characterize their response to abscisic acid (ABA) in culture, describe other mutants with related phenotypes, and summarize studies with double mutants. Our results indicate that immature embryos precociously enter a germination pathway after the torpedo stage of development and then acquire characteristics normally restricted to vegetative parts of the plant. In contrast to other viviparous mutants of maize (vp1) and Arabidopsis (abi3) that produce ABA-insensitive embryos, immature lec embryos are sensitive to ABA in culture. ABA is therefore necessary but not sufficient for embryonic maturation in Arabidopsis. Three other mutants that produce trichomes on cotyledons following precocious germination in culture are described. One mutant is allelic to lec1, another is a fusca mutant (fus3), and the third defines a new locus (lec2). Mutant embryos differ in morphology, desiccation tolerance, pattern of anthocyanin accumulation, presence of storage materials, size and frequency of trichomes on cotyledons, and timing of precocious germination in culture. The leafy cotyledon phenotype has therefore allowed the identification of an important network of regulatory genes with overlapping functions during embryonic maturation in Arabidopsis.

A FUSCA gene of Arabidopsis encodes a novel protein essential for plant development.

Arabidopsis fusca mutants display striking purple coloration due to anthocyanin accumulation in their cotyledons. We describe six recessive fusca mutants isolated from Agrobacterium-transformed Arabidopsis families. These mutants first become defective during embryogenesis and exhibit limited seedling development. Double mutant constructs revealed that developmental defects were not simply a consequence of anthocyanin accumulation. fusca seedlings showed altered responses to several environmental and endogenous factors. Allelism tests established that three fusca loci are represented by mutants previously described as defective in light-regulated responses. To study the molecular basis of the fusca phenotype, we cloned the FUS6 gene. FUS6 encodes a novel protein that is hydrophilic, alpha-helical, and contains potential protein kinase C phosphorylation sites. The FUSCA proteins appear to act in a network of signal transduction pathways critical for plant development.

Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis.

Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.

A Homoeotic Mutant of Arabidopsis thaliana with Leafy Cotyledons.

Cotyledons are specialized leaves produced during plant embryogenesis. Cotyledons and leaves typically differ in morphology, ultrastructure, and patterns of gene expression. The leafy cotyledon (Iec) mutant of Arabidopsis thaliana fails to maintain this distinction between embryonic and vegetative patterns of plant development. Mutant embryos are phenotypically abnormal, occasionally viviparous, and intolerant of desiccation. Mutant cotyledons produce trichomes characteristic of leaves, lack embryo-specific protein bodies, and exhibit a vascular pattern intermediate between that of leaves and cotyledons. These results suggest that lec cotyledons are partially transformed into leaves and that the wild-type gene (LEC) functions to activate a wide range of embryo-specific pathways in higher plants.

Mapping genes essential for embryo development in Arabidopsis thaliana.

We 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 bio1 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.

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.

Arrested Embryos from the bio1 Auxotroph of Arabidopsis thaliana Contain Reduced Levels of Biotin.

The bio1 auxotroph of Arabidopsis thaliana is a recessive embryonic lethal that forms normal plants in the presence of biotin. The purpose of this study was to determine whether aborted seeds produced by heterozygous plants grown without vitamin supplements contained reduced levels of biotin. Two methods were used to determine the biotin content of mutant and wild-type tissues: streptavidin binding in microtiter plates and growth of the biotin-requiring bacterium Lactobacillus plantarum. Total biotin was measured in extracts prepared from immature seeds prior to desiccation. Aborted seeds produced by heterozygous (bio1/BIO1) plants contained some biotin in the maternal seed coat but virtually no detectable biotin in the arrested embryo. This lack of biotin was not observed in arrested embryos from other mutants with similar patterns of abnormal development. These results are consistent with the model that bio1 tissues are defective in biotin synthesis. The alternative model of increased degradation is inconsistent with the recessive nature of the mutation and the ability of rescued plants to continue growing for several weeks following removal of supplemental biotin.