VACUOLELESS1 is an essential gene required for vacuole formation and morphogenesis in Arabidopsis.

Most plant cells are characterized by the presence of a large central vacuole that in differentiated cells accounts for more than 90% of the total volume. We have undertaken a genetic screen to look for mutants that are affected in the formation of vacuoles in plants. In this study, we report that inactivation of the Arabidopsis gene VACUOLELESS1 (VCL1) blocks vacuole formation and alters the pattern of cell division orientation and cell elongation in the embryo. Consistent with a role in vacuole biogenesis, we show that VCL1 encodes the Arabidopsis ortholog of yeast Vps16p. In contrast to yeast mutants that lack a vacuolar compartment but are viable and morphologically normal, loss of the plant vacuole leads to aberrant morphogenesis and embryonic lethality.

Dissecting desaturation: plants prove advantageous.

Fatty acid desaturases play important roles in controlling the physical properties o f membranes and in the synthesis of signal molecules such as prostaglandins and pheromones. Most desaturases are membrane proteins that have been recalcitrant to characterization by conventional biochemical methods. Only one enzyme o f this class has been characterized from animals or fungi. In this context, plants have proved to be useful sources of experimental materials. Substantial progress has been made in characterizing and manipulating nine classes of desaturases that control the fatty acid composition o f both plant membranes and plant storage lipids, which account for approximately -30% of the calories in the human diet.

Genomics. Plant biology in 2010.

The completion of the Arabidopsis sequence will be followed by a new ten-year project that will determine the function of all angiosperm genes. Funding for the U.S. component of this multinational project will originate from a new initiative from the U.S. National Science Foundation called the 2010 Project. Progress toward completion of this ambitious project will necessitate significant changes in how the plant biology community selects and approaches research objectives. The plan envisions that the project will facilitate the development of a computational model of a virtual plant that will allow predictive queries about basic mechanisms underlying plant growth and development.

Plant lipids: metabolism, mutants, and membranes.

The mechanisms that regulate plant lipid metabolism determine the dietary and industrial value of storage oils found in economically important species and may control the ability of many plants to survive exposure to temperature extremes. Many of the problems researchers have in defining the pathways, enzymes, and genes involved in plant lipid metabolism appear to be amenable to analysis by genetic approaches. Mutants with alterations in membrane lipid composition have also been used to study the structural and adaptive roles of lipids. The application of genetic engineering methods affords opportunities for researchers to apply knowledge gained about plant lipid metabolism toward enhanced use of plant oils as abundant and renewable sources of reduced carbon.

Plant functional genomics.

Nucleotide sequencing of the Arabidopsis genome is nearing completion, sequencing of the rice genome has begun, and large amounts of expressed sequence tag information are being obtained for many other plants. There are many opportunities to use this wealth of sequence information to accelerate progress toward a comprehensive understanding of the genetic mechanisms that control plant growth and development and responses to the environment.

A mutant of Arabidopsis lacking a chloroplast-specific lipid.

In order to investigate the functional significance of membrane lipid unsaturation, we have isolated a series of mutants of Arabidopsis thaliana which are deficient in particular membrane fatty acids. The first of these mutants completely lacks Delta3-trans-hexadecenoate, an acyl group that until now has been thought to play an important role in the structure or function of thylakoid membranes in photosynthetic eukaryotes. The apparent absence of any marked physiological effect of the mutation illustrates the potential of this approach to the analysis of membrane structure and function.

Catalytic plasticity of fatty acid modification enzymes underlying chemical diversity of plant lipids.

Higher plants exhibit extensive diversity in the composition of seed storage fatty acids. This is largely due to the presence of various combinations of double or triple bonds and hydroxyl or epoxy groups, which are synthesized by a family of structurally similar enzymes. As few as four amino acid substitutions can convert an oleate 12-desaturase to a hydroxylase and as few as six result in conversion of a hydroxylase to a desaturase. These results illustrate how catalytic plasticity of these diiron enzymes has contributed to the evolution of the chemical diversity found in higher plants.

Altered growth and cell walls in a fucose-deficient mutant of Arabidopsis.

A biochemical screening procedure was developed to identify mutants of Arabidopsis thaliana in which the polysaccharide composition of the cell wall was altered. Over 5000 ethyl methanesulfonate-mutagenized plants were analyzed by this method, leading to the identification of 38 mutant lines. One complementation group of mutants was completely deficient in l-fucose, a constituent of pectic and hemicellulosic polysaccharides. These mutant plants were dwarfed in growth habit, and their cell walls were considerably more fragile than normal.

Cellular differentiation regulated by gibberellin in the Arabidopsis thaliana pickle mutant.

The plant growth regulator gibberellin (GA) has a profound effect on shoot development and promotes developmental transitions such as flowering. Little is known about any analogous effect GA might have on root development. In a screen for mutants, Arabidopsis plants carrying a mutation designated pickle (pkl) were isolated in which the primary root meristem retained characteristics of embryonic tissue. Expression of this aberrant differentiation state was suppressed by GA. Root tissue from plants carrying the pkl mutation spontaneously regenerated new embryos and plants.

Insensitivity to Ethylene Conferred by a Dominant Mutation in Arabidopsis thaliana.

Ethylene influences a number of developmental processes and responses to stress in higher plants. The molecular basis for the action of ethylene was investigated in mutants of Arabidopsis thaliana that have altered responses to ethylene. One mutant line, which has a dominant mutation at a locus designated etr, lacks a number of responses to ethylene that are present in the wild-type plant. These include inhibition of cell elongation, promotion of seed germination, enhancement of peroxidase activity, acceleration of leaf senescence, and feedback suppression of ethylene synthesis by ethylene. These diverse responses, which occur in different tissues of Arabidopsis, appear to share some common element in their transduction pathways-for example, a single receptor for ethylene. Results of ethylene binding experiments in vivo indicate that this receptor may be affected by the etr mutation.

Polyhydroxybutyrate, a biodegradable thermoplastic, produced in transgenic plants.

Polyhydroxybutyrate (PHB), a high molecular weight polyester, is accumulated as a storage carbon in many species of bacteria and is a biodegradable thermoplastic. To produce PHB by genetic engineering in plants, genes from the bacterium Alcaligenes eutrophus that encoded the two enzymes required to convert acetoacetyl-coenzyme A to PHB were placed under transcriptional control of the cauliflower mosaic virus 35S promoter and introduced into Arabidopsis thaliana. Transgenic plant lines that contained both genes accumulated PHB as electron-lucent granules in the cytoplasm, nucleus, and vacuole; the size and appearance of these granules were similar to the PHB granules that accumulate in bacteria.

Map-based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis.

A gene from the flowering plant Arabidopsis thaliana that encodes an omega-3 desaturase was cloned on the basis of the genetic map position of a mutation affecting membrane and storage lipid fatty acid composition. Yeast artificial chromosomes covering the genetic locus were identified and used to probe a seed complementary DNA library. A complementary DNA clone for the desaturase was identified and introduced into roots of both wild-type and mutant plants by Ti plasmid-mediated transformation. Transgenic tissues of both mutant and wild-type plants had significantly increased amounts of the fatty acid produced by this desaturase.

Cloning in silico.

Cellulose is a major component of plant cell walls, but identification of the enzymes that synthesize it has proven difficult. Now, however, several candidate proteins with sequence homology to bacterial cellulose synthases have been identified by partial sequencing of anonymous cDNA clones from cotton fibers.

Genetic Control of Root Hair Development in Arabidopsis thaliana.

Visual examination of roots from 12,000 mutagenized Arabidopsis seedlings has led to the identification of more than 40 mutants impaired in root hair morphogenesis. Mutants from four phenotypic classes have been characterized in detail, and genetic tests show that these result from single nuclear recessive mutations in four different genes designated RHD1, RHD2, RHD3, and RHD4. The phenotypic analysis of the mutants and homozygous double mutants has led to a proposed model for root hair development and the stages at which the genes are normally required. The RHD1 gene product appears to be necessary for proper initiation of root hairs, whereas the RHD2, RHD3, and RHD4 gene products are required for normal hair elongation. These results demonstrate that root hair development in Arabidopsis is amenable to genetic dissection and should prove to be a useful model system to study the molecular mechanisms governing cell differentiation in plants.

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).

Preliminary crystallographic data for stearoyl-acyl carrier protein desaturase from castor seed.

Recombinant stearoyl-acyl carrier protein desaturase (EC 1.14.99.6) from castor seed has been crystallized with polyethylene glycol 8000 as precipitant. The crystals are orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 81.3, b = 146.4 and c = 197.7 A. The observed diffraction pattern extends to at least 2.5 A resolution. Rotation function calculations indicate a non-crystallographic 3-fold rotation axis parallel to the crystallographic a-axis. Perpendicular to this axis, 2-fold rotation axes were found at 30 degrees intervals, i.e. maxima at kappa = 180 degrees, phi = 90 degrees and omega = 30 degrees and 60 degrees, respectively. Together with the packing density of the crystals (Vm = 2.4 A3/Da for n = 6), these results suggest, that the crystal asymmetric unit most likely contains a hexamer of desaturase subunits.

Phenotypic Suppression of the Gibberellin-Insensitive Mutant (gai) of Arabidopsis.

The semidominant gibberellin-insensitive (gai) mutant of Arabidopsis thaliana shows impairment in multiple responses to the plant hormone gibberellin A3, which include effects on seed germination, stem elongation, apical dominance, and rapid flowering in short days. Results presented here show that the gai mutation also interferes with development of fertile flowers in continuous light. Mu-tagenesis of the gai mutant resulted in recovery of 17 independent mutants in which the gibberellin-insensitive phenotype is partially or completely suppressed. Sixteen of the suppressor mutations act semidominantly to restore gibberellin responsiveness. One representative of this class, the gar1 mutation, could not be genetically separated from the gai locus and is proposed to cause inactivation of the gai gene. The exceptional gar2 mutation partially suppresses the gai phenotype, is completely dominant, and is not linked to the gai locus. The gar2 mutation may define a new gene involved in gibberellin signaling. A recessive allele of the spindly (SPY) locus, spy-5, was also found to partially suppress the gai mutant phenotype.

A Mutation at the fad8 Locus of Arabidopsis Identifies a Second Chloroplast [omega]-3 Desaturase.

Two independently isolated mutations at the fad7 locus in Arabidopsis produced plants with a temperature-conditional phenotype. Leaves of fad7 mutants grown at 28[deg]C contained less than 30% of wild-type levels of trienoic fatty acids (16:3 plus 18:3) compared with more than 70% of wild-type levels for plants grown at 15[deg]C. Screening of an M2 population derived from the fad7-1 line led to the identification of a line, SH1, in which the proportion of trienoic acids was much less than in fad7 plants. The segregation pattern of F2 progeny from a cross between SH1 and wild type indicated that the additional fatty acid mutation in SH1 is at a new locus, designated fad8. In a genetic background that was wild type at the FAD7 locus, the fad8 mutation had no detectable effect on overall leaf fatty acid composition irrespective of the temperature at which plants were grown. However, fatty acid analyses of individual leaf lipids revealed small decreases in the levels of 18:3 in two chloroplast lipids. In fad8 plants grown at 22[deg]C, phospha-tidylglycerol contained 22.5% 18:3 compared with 33.5% in wild-type Arabidopsis. For sulfoquinovosyldiacylglycerol, the values were 31.4 and 44.5%, respectively. Together with information from studies of the cloned FAD8 gene (S. Gibson, V. Arondel, K. Iba, C. Somerville [1994] Plant Physiol 106: 1615-1621), these results indicate that the FAD8 locus encodes a chloroplast-localized 16:2/18:2 desaturase that has a substrate specificity similar to the FAD7 gene product but that is induced by low temperature.

Expressed Sequence Tags from Developing Castor Seeds.

To expand the availability of genes encoding enzymes and structural proteins associated with storage lipid synthesis and deposition, partial nucleotide sequences, or expressed sequence tags (ESTs), were obtained for 743 cDNA clones derived from developing seeds of castor (Ricinus communis L.). Enrichment for seed-specific cDNA clones was obtained by selecting clones that did not detectably hybridize to first-strand cDNA from leaf mRNA. Similarly, clones that hybridized to storage proteins or other highly abundant mRNA species from developing seeds were selected against. To enrich for endomembrane-associated proteins, some clones were selected for sequencing by immunological screening with antibodies prepared against partially purified endoplasmic reticulum membranes. Comparison of the deduced amino acid sequences of the ESTs with the public data bases resulted in the assignment of putative identities of 49% of the clones selected by differential hybridization and 71% of the clones selected by immunological screening. Open reading frames in 100 of the ESTs exhibited higher homology to 78 different nonplant gene products than to any previously known plant gene product.

Genetic engineering of commercially useful biosynthetic pathways in transgenic plants.

In many economically important plant species, the chemical composition of one or more non-protein compounds determines the value of the plant and may have an important role in protecting the plant from environmental stress, including pests, drought, salt, temperature and light. A number of potential opportunities exist whereby the range or amount of such valuable compounds can be increased by genetic engineering.