The Arabidopsis BELL1 and KNOX TALE homeodomain proteins interact through a domain conserved between plants and animals.

Interactions between TALE (three-amino acid loop extension) homeodomain proteins play important roles in the development of both fungi and animals. Although in plants, two different subclasses of TALE proteins include important developmental regulators, the existence of interactions between plant TALE proteins has remained unexplored. We have used the yeast two-hybrid system to demonstrate that the Arabidopsis BELL1 (BEL1) homeodomain protein can selectively heterodimerize with specific KNAT homeodomain proteins. Interaction is mediated by BEL1 sequences N terminal to the homeodomain and KNAT sequences including the MEINOX domain. These findings validate the hypothesis that the MEINOX domain has been conserved between plants and animals as an interaction domain for developmental regulators. In yeast, BEL1 and KNAT proteins can activate transcription only as a heterodimeric complex, suggesting a role for such complexes in planta. Finally, overlapping patterns of BEL1 and SHOOT MERISTEMLESS (STM) expression within the inflorescence meristem suggest a role for the BEL1-STM complex in maintaining the indeterminacy of the inflorescence meristem.

Isolation and characterization of mutants defective in seed coat mucilage secretory cell development in Arabidopsis.

In Arabidopsis, fertilization induces the epidermal cells of the outer ovule integument to differentiate into a specialized seed coat cell type producing extracellular pectinaceous mucilage and a volcano-shaped secondary cell wall. Differentiation involves a regulated series of cytological events including growth, cytoplasmic rearrangement, mucilage synthesis, and secondary cell wall production. We have tested the potential of Arabidopsis seed coat epidermal cells as a model system for the genetic analysis of these processes. A screen for mutants defective in seed mucilage identified five novel genes (MUCILAGE-MODIFIED [MUM]1-5). The seed coat development of these mutants, and that of three previously identified ones (TRANSPARENT TESTA GLABRA1, GLABRA2, and APETALA2) were characterized. Our results show that the genes identified define several events in seed coat differentiation. Although APETALA2 is needed for differentiation of both outer layers of the seed coat, TRANSPARENT TESTA GLABRA1, GLABRA2, and MUM4 are required for complete mucilage synthesis and cytoplasmic rearrangement. MUM3 and MUM5 may be involved in the regulation of mucilage composition, whereas MUM1 and MUM2 appear to play novel roles in post-synthesis cell wall modifications necessary for mucilage extrusion.

Differentiation of mucilage secretory cells of the Arabidopsis seed coat.

In some plant species, including Arabidopsis, fertilization induces the epidermal cells of the outer ovule integument to differentiate into a specialized seed coat cell type with a unique morphology and containing large quantities of polysaccharide mucilage (pectin). Such seed coat mucilage cells are necessary for neither viability nor germination under normal laboratory conditions. Thus, the Arabidopsis seed coat offers a unique system with which to use genetics to identify genes controlling cell morphogenesis and complex polysaccharide biosynthesis and secretion. As a first step in the application of this system, we have used microscopy to investigate the structure and differentiation of Arabidopsis seed coat mucilage cells, including cell morphogenesis and the synthesis, secretion, and extrusion of mucilage. During seed coat development in Arabidopsis, the epidermal cells of the outer ovule integument grow and differentiate into cells that produce large quantities of mucilage between the primary cell wall and plasma membrane. Concurrent with mucilage production, the cytoplasm is shaped into a column in the center of the cell. Following mucilage secretion the cytoplasmic column is surrounded by a secondary cell wall to form a structure known as the columella. Thus, differentiation of the seed coat mucilage cells involves a highly regulated series of events including growth, morphogenesis, mucilage biosynthesis and secretion, and secondary cell wall synthesis.

The UNUSUAL FLORAL ORGANS gene of Arabidopsis thaliana is an F-box protein required for normal patterning and growth in the floral meristem.

Genetic and molecular studies have suggested that the UNUSUAL FLORAL ORGANS (UFO) gene, from Arabidopsis thaliana, is expressed in all shoot apical meristems, and is involved in the regulation of a complex set of developmental events during floral development, including floral meristem and floral organ identity. Results from in situ hybridization using genes expressed early in floral development as probes indicate that UFO controls growth of young floral primordia. Transgenic constructs were used to provide evidence that UFO regulates floral organ identity by activating or maintaining transcription of the class B organ-identity gene APETALA 3, but not PISTILLATA. In an attempt to understand the biochemical mode of action of the UFO gene product, we show here that UFO is an F-box protein that interacts with Arabidopsis SKP1-like proteins, both in the yeast two-hybrid system and in vitro. In yeast and other organisms both F-box proteins and SKP1 homologues are subunits of specific ubiquitin E3 enzyme complexes that target specific proteins for degradation. The protein selected for degradation by the complex is specified by the F-box proteins. It is therefore possible that the role of UFO is to target for degradation specific proteins controlling normal growth patterns in the floral primordia, as well as proteins that negatively regulate APETALA 3 transcription.

BELL1 and AGAMOUS genes promote ovule identity in Arabidopsis thaliana.

Molecular and genetic analyses have demonstrated that the Arabidopsis thaliana gene BELL1 (BEL1) is required for proper morphogenesis of the ovule integuments. Several lines of evidence suggest that BEL1 may act, at least in part, to repress the function of the organ identity gene AGAMOUS (AG) during ovule development. To study the relative roles of BEL1 and AG, plants homozygous for ag, bel1 or both were constructed in an ap2 mutant background where ovules form even in the absence of AG function. The loss of either BEL1 or AG led to a decrease in the number of mature ovules, accompanied by an increase in primordial outgrowths. These data suggest that BEL1 and AG gene products act early in ovule development in a partially redundant manner to direct ovule identity. Development of the abnormal integuments characteristic of the Bel1- mutant phenotype was found to be dependent on AG function. Finally, BEL1 appears to be required for embryo sac development independent of both other aspects of ovule morphogenesis and AG function. This study therefore suggests that both BEL1 and AG are required for several distinct aspects of ovule morphogenesis.

Different roles of flowering-time genes in the activation of floral initiation genes in Arabidopsis.

We have analyzed double mutants that combine late-flowering mutations at four flowering-time loci (FVE, FPA, FWA, and FT) with mutations at the LEAFY (LFY), APETALA1 (AP1), and TERMINAL FLOWER1 (TFL1) loci involved in the floral initiation process (FLIP). Double mutants between ft-1 or fwa-1 and lfy-6 completely lack flowerlike structures, indicating that both FWA and FT act redundantly with LFY to control AP1. Moreover, the phenotypes of ft-1 ap1-1 and fwa-1 ap1-1 double mutants are reminiscent of the phenotype of ap1-1 cal-1 double mutants, suggesting that FWA and FT could also be involved in the control of other FLIP genes. Such extreme phenotypes were not observed in double mutants between fve-2 or fpa-1 and lfy-6 ap1-1. Each of these showed a phenotype similar to that of ap1-1 or lfy-6 mutants grown under noninductive photoperiods, suggesting a redundant interaction with FLIP genes. Finally, the phenotype of double mutants combining the late-flowering mutations with tfl1-2 were also consistent with the different roles of flowering-time genes.

Divergence of function and regulation of class B floral organ identity genes.

Regulatory mechanisms controlling basic aspects of floral morphogenesis seem to be highly conserved among plant species. The class B organ identity genes, which are required to establish the identity of organs in the second (petals) and third (stamens) floral whorls, are a good example of such conservation. This work compares the function of two similar class B genes in the same genetic background. The DEFICIENS (DEF) gene from Antirrhinum, including its promoter, was transformed into Arabidopsis and compared in function and expression with the Arabidopsis class B genes APETALA3 (AP3) and PISTILLATA (PI). The DEF gene was expressed in the second, third, and fourth whorls, as was PI. Functionally, DEF could replace AP3 in making petals and stamens. The DEF gene's AP3-like function and PI-like expression caused transformation of fourth-whorl carpels to stamens. Like AP3, all aspects of DEF function in Arabidopsis required a functional PI protein. Surprisingly, DEF could not replace the AP3 protein in properly maintaining AP3 transcripts (autoregulation). Our data allow us to revise the current model for class B autoregulation and propose a hypothesis for the evolution of class B gene expression in dicotyledonous plants.

Non-cell-autonomous function of the Antirrhinum floral homeotic proteins DEFICIENS and GLOBOSA is exerted by their polar cell-to-cell trafficking.

In Antirrhinum majus, petal and stamen organ identity is controlled by two MADS-box transcription factors, DEFICIENS and GLOBOSA. Mutations in either of these genes result in the replacement of petals by sepaloid organs and stamens by carpelloid organs. Somatically stable def and glo periclinal chimeras, generated by transposon excision events, were used to study the non-cell-autonomous functions of these two MADS-box proteins. Two morphologically distinct types of chimeras were analysed using genetic, morphological and molecular techniques. Restoration of DEF expression in the L1 cell layer results in the reestablishment of DEF and GLO functions in L1-derived cells only; inner layer cells retain their mutant sepaloid features. Nevertheless, this activity is sufficient to allow the expansion of petal lobes, highlighting the role of DEF in the stimulation of cell proliferation and/or cell shape and elongation when expressed in the L1 layer. Establishment of DEF or GLO expression in L2 and L3 cell layers is accompanied by the recovery of petaloid identity of the epidermal cells but it is insufficient to allow petal lobe expansion. We show by in situ immunolocalisation that the non-cell-autonomy is due to direct trafficking of DEF and GLO proteins from the inner layer to the epidermal cells. At least for DEF, this movement appears to be polar since DEF acts cell-autonomously when expressed in the L1 cell layer. Furthermore, the petaloid revertant sectors observed on second whorl mutant organs and the mutant margins of petals of L2L3 chimeras suggest that DEF and GLO intradermal movement is limited. This restriction may reflect the difference in the regulation of primary plasmodesmata connecting cells from the same layer and secondary plasmodesmata connecting cells from different layers. We propose that control of intradermal trafficking of DEF and GLO could play a role in maintaining of the boundaries of their expression domains.

The BELL1 gene encodes a homeodomain protein involved in pattern formation in the Arabidopsis ovule primordium.

Ovule development in Arabidopsis involves the formation of three morphologically defined proximal-distal pattern elements. Integuments arise from the central pattern element. Analysis of Bell 1 (Bel 1) mutant ovules indicated that BEL1 was required for integument development. Cloning of the BEL1 locus reveals that it encodes a homeodomain transcription factor. Prior to integument initiation, BEL1 RNA localizes to the central domain, providing molecular evidence for a central pattern element. Therefore, proximal-distal patterning of the ovule involves the regulated expression of the BEL1 gene that controls integument morphogenesis. A model for BEL1 function is evaluated with regard to new data showing the expression pattern of the floral homeotic gene AGAMOUS (AG) early in wild-type and BEL1 ovule development.

UNUSUAL FLORAL ORGANS Controls Meristem Identity and Organ Primordia Fate in Arabidopsis.

A novel gene that is involved in regulating flower initiation and development has been identified in Arabidopsis. This gene has been designated UNUSUAL FLORAL ORGANS (UFO), with five corresponding nuclear recessive alleles designated ufo[middot]1 to ufo[middot]5. Under short day-length conditions, ufo homozygotes generate more coflorescences than do the wild type, and coflorescences often appear apical to the first floral shoot, resulting in a period of inflorescence development in which regions of floral and coflorescence shoots are produced alternately. ufo enhances the phenotype of weak leafy alleles, and the double mutant Ufo-1 Apetala1-1 produces only coflorescence-like shoots, suggesting that these two genes control different aspects of floral initiation. Floral development was also altered in Ufo plants. Ufo flowers have an altered organ number in all whorls, and organs in the first, second, and third whorls exhibit variable homeotic transformations. Ufo single and double mutant phenotypes suggest that the floral changes result from reduction in class B floral homeotic gene expression and fluctuations in the expression boundaries of class C function and FLO10. Surprisingly, in situ hybridization analysis revealed no obvious differences in expression pattern or level in developing Ufo flowers compared with that of the wild type for any class B or C gene studied. We propose that UFO acts in concert with known floral initiation genes and regulates the domains of floral homeotic gene function.

Parallels between UNUSUAL FLORAL ORGANS and FIMBRIATA, genes controlling flower development in Arabidopsis and Antirrhinum.

The unusual floral organs (ufo) mutant of Arabidopsis has flowers with variable homeotic organ transformations and inflorescence-like characteristics. To determine the relationship between UFO and previously characterized meristem and organ identity genes, we cloned UFO and determined its expression pattern. The UFO gene shows extensive homology with FIMBRIATA (FIM), a gene mediating between meristem and organ identity genes in Antirrhinum. All three UFO mutant alleles that we sequenced are predicted to produce truncated proteins. UFO transcripts were first detected in early floral meristems, before organ identity genes had been activated. At later developmental stages, UFO expression is restricted to the junction between sepal and petal primordia. Phenotypic, genetic, and expression pattern comparisons between UFO and FIM suggest that they are cognate homologs and play a similar role in mediating between meristem and organ identity genes. However, some differences in the functions and genetic interactions of UFO and FIM were apparent, indicating that changes in partially redundant pathways have occurred during the evolutionary divergence of Arabidopsis and Antirrhinum.

In planta transformation of Arabidopsis thaliana.

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7-8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny.(ABSTRACT TRUNCATED AT 250 WORDS)

Intragenic recombination in the CSR1 locus of Arabidopsis.

Four classes of herbicides are known to inhibit plant acetolactate synthase (ALS). In Arabidopsis, ALS is encoded by a single gene, CSR1. The dominant csr1-1 allele encodes an ALS resistant to chlorsulfuron and triazolopyrimidine sulfonamide while the dominant csr1-2 allele encodes an ALS resistant to imazapyr and pyrimidyl-oxy-benzoate. The molecular distance between the point mutations in csr1-1 and csr1-2 is 1369 bp. Here we used multiherbicide resistance as a stringent selection to measure the intragenic recombination frequency between these two point mutations. We found this frequency to be 0.008 +/- 0.0028. The recombinant multiherbicide-resistant allele, csr1-4, provides an ideal marker for plant genetic transformation.

Homeotic Transformation of Ovules into Carpel-like Structures in Arabidopsis.

Ovules are specialized reproductive organs that develop within the carpels of higher plants. In Arabidopsis, mutations in two genes, BELL1 (BEL1) and APETALA2 (AP2), disrupt ovule development. In Bel1 ovules, the inner integument fails to form, the outer integument develops abnormally, and the embryo sac arrests at a late stage of megagametogenesis. During later stages of ovule development, cells of the outer integument of a Bel1 ovule sometimes develop into a carpel-like structure with stigmatic papillae and second-order ovules. The frequency of carpel-like structures was highest when plants were grown under conditions that normally induced flowering and was correlated with ectopic expression in the ovule of AGAMOUS (AG), an organ-identity gene required for carpel formation. Together, these results suggested that BEL1 negatively regulates AG late in ovule development. Likewise, mutants homozygous for the strong AP2 allele ap2-6 sometimes displayed structures with carpel-like features in place of ovules. However, such abnormal Ap2 ovules are much less ovulelike in morphology and form earlier than the Bel1 carpel-like structures. Because one role of the AP2 gene is to negatively regulate AG expression early in flower development, it is possible that AP2 works in a similar manner in the ovule. A novel ovule phenotype observed in Bel1/Ap2-6 double mutants suggested that BEL1 and AP2 genes function independently during ovule development.

Molecular Basis of Imidazolinone Herbicide Resistance in Arabidopsis thaliana var Columbia.

Acetolactate synthase (ALS), the first enzyme in the biosynthetic pathway of leucine, isoleucine, and valine, is inhibited by imidazolinone herbicides. To understand the molecular basis of imidazolinone resistance, we isolated the ALS gene from an imazapyr-resistant mutant GH90 of Arabidopsis thaliana. DNA sequence analysis of the mutant ALS gene demonstrated a single-point mutation from G to A at nucleotide 1958 of the ALS-coding sequence. This would result in Ser to Asn substitution at residue 653 near the carboxyl terminal of the matured ALS. The mutant ALS gene was introduced into tobacco using Agrobacterium-mediated transformation. Imidazolinone-resistant growth of transformed calli and leaves of transgenic plants was 100-fold greater than that of nontransformed control plants. The relative levels of imidazolinone-resistant ALS activity correlated with the amount of herbicide-resistant growth in the leaves of transgenic plants. Southern hybridization analysis confirmed the existence of transferred ALS gene in the transformant showing high imazapyr resistance. The results demonstrate that the mutant ALS gene confers resistance to imidazolinone herbicides. This is the first report, to our knowledge, of the molecular basis of imidazolinone resistance in plants.

The FLO10 Gene Product Regulates the Expression Domain of Homeotic Genes AP3 and PI in Arabidopsis Flowers.

We describe a novel mutant of Arabidopsis, Flo10, which is the result of a recessive allele, flo10, in the nuclear gene FLO10. The first three organ whorls (sepals, petals, and stamens) of Flo10 flowers are normal, but the fourth, gynoecial whorl is replaced by two to eight stamens or stamen-carpel intermediate organs. Studies of ontogeny suggest that the position of the first six of these fourth-whorl organs often resembles that of the wild-type third-whorl organs. To determine the interaction of the FLO10 gene with the floral organ homeotic genes APETALA2 (AP2), PISTILLATA (PI), AP3, and AGAMOUS (AG), we generated lines homozygous for flo10 and heterozygous or homozygous for a recessive allele of the homeotic genes. On the basis of our data, we suggest that FLO10 functions to prevent the expression of the AP3/PI developmental pathway in the gynoecial (fourth) whorl.

Biochemical Genetics of Plant Secondary Metabolites in Arabidopsis thaliana: The Glucosinolates.

Mutants of Arabidopsis thaliana with a glucosinolate content different from wild type were isolated by screening a mutagenized population of plants. Six mutants were detected out of a population of 1200 screened. One of these mutants, TU1, was analyzed in detail. Leaf and seed tissues of line TU1 lack or have reduced amounts of many of the aliphatic glucosinolates found in the wild type due to a recessive allele, gsm1, of a single nuclear gene, GSM1. The seed phenotype is inherited as a maternal effect suggesting that the embryo is dependent on the maternal tissue for its glucosinolates. Experiments involving feeding of (14)C-labeled intermediates suggested that the gsm1 allele results in a metabolic block which decreases the availability of several amino acid substrates required for glucosinolate biosynthesis: 2-amino-6-methylthiohexanoic acid, 2-amino-7-methylthioheptanoic acid, and 2-amino-8-methylthiooctanoic acid. The mutation does not result in any obvious changes in morphology or growth rate. A pathway for the biosynthesis of glucosinolates in A. thaliana is proposed.

LEAFY, a Homeotic Gene That Regulates Inflorescence Development in Arabidopsis.

Variation in plant shoot structure may be described as occurring through changes within a basic unit, the metamer. Using this terminology, the apical meristem of Arabidopsis produces three metameric types sequentially: type 1, rosette; type 2, coflorescence-bearing with bract; and type 3, flower-bearing without bract. We describe a mutant of Arabidopsis, Leafy, homozygous for a recessive allele of a nuclear gene LEAFY (LFY), that has an inflorescence composed only of type 2-like metamers. These data suggest that the LFY gene is required for the development of type 3 metamers and that the transition from type 2 to type 3 metamers is a developmental step distinct from that between vegetative and reproductive growth (type 1 to type 2 metamers). Results from double mutant analysis, showing that lfy-1 is epistatic to the floral organ homeotic gene ap2-6, are consistent with the hypothesis that a functional LFY gene is necessary for the expression of downstream genes controlling floral organ identity.