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.

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.

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.

AP2 Gene Determines the Identity of Perianth Organs in Flowers of Arabidopsis thaliana.

We have examined the floral morphology and ontogeny of three mutants of Arabidopsis thaliana, Ap2-5, Ap2-6, and Ap2-7, that exhibit homeotic changes of the perianth organs because of single recessive mutations in the AP2 gene. Homeotic conversions observed are: sepals to carpels in all three mutants, petals to stamens in Ap2-5, and petals to carpels in Ap2-6. Our analysis of these mutants suggests that the AP2 gene is required early in floral development to direct primordia of the first and second whorls to develop as perianth rather than as reproductive organs. In addition, our results support one of the two conflicting hypotheses concerning the structures of the calyx and the gynoecium in the Brassicaceae.

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.

Transcription initiation sites of the leucine operons of Salmonella typhimurium and Escherichia coli.

Evidence for a transcription attenuation site downstream from the leu promoter was obtained by transcription experiments in vitro. Most transcription initiated in vitro from leuP is terminated prematurely, resulting in the synthesis of a 160 nucleotide leader RNA. We define here the point at which transcription is initiated in vitro and in vivo and demonstrate that the site of premature termination is between the promoter and the first structural gene (leuA). Additional nucleotide sequences are presented that extend the known sequence 200 base-pairs upstream and 300 base-pairs downstream from leuP. The location of the promoter-proximal end of cistron leuA was deduced by comparing nucleotide sequence data with the sequence of the ten amino acids at the N-terminus of alpha-isopropylmalate synthase. To facilitate the isolation of quantities of material for sequencing experiments, the enzyme was isolated from a plasmid-containing strain, CV605, grown under conditions of leucine limitation. Under such conditions, about 20% of the total soluble protein of strain CV605 is alpha-isopropylmalate synthase and another 20% is beta-isopropylmalate dehydrogenase (leuB product).

Induction and segregation of chloroplast mutations in vegetative cell cultures of chlamydomonas reinhardtii.

The single chloroplast of the alga Chlamydomonas reinhardtii contains at least 100 copies of the chloroplast chromosome. It is not known how the chloroplast (or cell) becomes homoplasmic for a mutation that arises in one of these copies. Under suitable selection conditions, clones with chloroplast mutations for streptomycin resistance induced by methyl methanesulfonate can be recovered with direct plating after mutagenesis. Using an adaptation of the Luria-Delbrück fluctuation test, mutagenized cultures grown on nonselective liquid medium for seven to nine doublings show negligible proliferation of cells capable of forming such mutant colonies. In contrast, cells among the same cultures with reduced nuclear mutations conferring streptomycin resistance reveal considerable clonal propagation prior to plating on selection medium. Reconstruction growth-rate experiments show no reduced growth of cells with chloroplast mutations relative to either wild-type cells or to those with nuclear mutations. We propose that newly arising chloroplast mutations and their copies are usually transmitted to only one daughter cell for several cell generations by reductional divisions of the chloroplast genome. In the absence of recombination and mixing, such a reductional partition of chloroplast alleles would readily permit the formation of homoplasmic lines without the need for selection.

A Mutation Causing Imidazolinone Resistance Maps to the Csr1 Locus of Arabidopsis thaliana.

A mutant of Arabidopsis thaliana, two hundred times more resistant to the imidazolinone herbicide imazapyr than wild-type plants, was isolated by direct selection of seedlings from a mutagenized population. Genetic analysis showed that resistance is due to a single dominant nuclear mutation that could not be separated by recombination from a mutation in the CSR1 gene encoding acetohydroxy acid synthase. Acetohydroxy acid synthase activity in extracts isolated from the mutant was 1000-fold more resistant to inhibition by imazapyr than that of the wild type. The resistant enzyme activity cosegregated with whole plant resistance. These data strongly suggest that the mutation is an allele of CSR1 encoding an imazapyr-resistant AHAS.

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.

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.

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.

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.

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.

A large family of bacterial activator proteins.

At least nine different bacterial proteins belong to the LysR family. The gene sequence for one of these proteins is presented here. Six others (Escherichia coli LysR, IlvY, CysB; Salmonella typhimurium MetR; Rhizobium NodD; and Enterobacter cloacae AmpR) are known to activate other genes. Based on sequence alignments, each member of this family is predicted to have a helix-turn-helix DNA binding motif near its amino terminus. The combined evidence indicates that all nine proteins are related by common ancestry, are similarly folded, and are not detectably related to other known bacterial regulatory proteins. The DNA database searching procedure and other methods used in this study should be useful in detecting other groups of related proteins.

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)

High A + T content conserved in DNA sequences upstream of leuABCD in Escherichia coli and Salmonella typhimurium.

The nucleotide sequence of over 800 base pairs of DNA upstream of leuP was determined for Escherichia coli and Salmonella typhimurium. In both of these enteric bacteria, approximately 500 base pairs of A + T-rich sequences separates leuP from an upstream open reading frame. Although these A + T-rich sequences share little homology, the distribution of A + T base pairs within the region is strikingly conserved. Deletion of the A + T-rich sequences upstream of the E. coli leu operon does not markedly affect the strength of the leu promoter in vivo.

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.

Unusual organization of the ilvIH promoter of Escherichia coli.

Analysis of plasmids containing ilvIH-galK fusions indicated that the Escherichia coli ilvIH promoter and sequences sufficient to cause leucine repression lie within 363 base pairs (bp) of ilvI. Experiments designed to locate the promoter and regulatory sequences more precisely gave the following results. The positions of the 5' endpoints of both unlabeled and pulse-labeled ilvIH mRNAs transcribed in vivo lie 30 bp upstream of ilvI. By contrast, the major in vitro RNA endpoints lie at positions further upstream. Several mutations which increase the expression of ilvIH lie 40 to 50 bp upstream of ilvI, within a putative promoter termed P1. Deletion of a 50-bp region immediately upstream of ilvI, which includes P1, resulted in the loss of all ilvIH promoter activity. Deletion of sequences more than 200 bp upstream of ilvI reduced ilvIH promoter activity by more than 80%. These results suggest that transcription of the ilvIH operon is initiated from promoter P1 but that sequences more than 200 bp upstream are required for optimal transcription of the operon.