Using the novel radioligand, [3H]-9' -nor-fusicoccin-8' -alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K+, Mg2+ -ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [3H]-9' -nor-fusicoccin-8' -alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (KD), KD1 = 1.5 nanomolar and KD2 = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrance of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 +/- 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin.

We have constructed a restriction fragment length polymorphism (RFLP) linkage map of the nuclear genome of the small flowering plant Arabidopsis thaliana. The map is based on the, meiotic segregation of both RFLP and morphological genetic markers from five independent crosses. The morphological markers on each of the five chromosomes were included in the crosses to allow alignment of the RFLP map with the established genetic map. The map contains 94 new randomly distributed molecular markers (nine identified cloned Arabidopsis genes and 85 genomic cosmid clones) that detect polymorphisms between the Landsberg erecta and Columbia races. In addition, 17 markers from an independently constructed RFLP map of the Arabidopsis genome [Chang, C., Bowman, J.L., DeJohn, A.W., Lander, E.S., and Meyerowitz, E.M. (1988). Proc. Natl. Acad. Sci. USA 85, 6856-6860] have been included to permit integration of the two RFLP maps.

Two mutants of Arabidopsis thaliana have been identified with decreased phototropism to 450-nanometer light. Fluence-response relationships for these strains (ZR8 and ZR19) to single and multiple flashes of light show thresholds, curve shapes, and fluence for maximum curvature in 'first positive' phototropism which are the same as those of the wild type. Similarly, there is no alteration from the wild type in the kinetics of curvature or in the optimum dark period separating sequential flashes in a multiple flash regimen. In addition, in both strains, gravitropism is decreased compared to the wild type by an amount which is comparable to the decrease in phototropism. Based on reciprocal backcrosses, it appears that the alteration is due to a recessive nuclear mutation. It is suggested that ZR8 and ZR19 represent alterations in some step analogous to an amplifier, downstream of the photoreceptor pigment, and common to both phototropism and gravitropism.

International audience | The gene family encoding the Arabidopsis thaliana translation elongation factor (EF-1 alpha) was analysed. This family contains four genes (A1-A4) organized in a similar manner in different varieties of Arabidopsis. Based upon both their physical separation and a comparison of their sequences, it is suggested that the A4 gene and the A1, A2, and A3 genes constitute two distinct subfamilies within the genome. By introducing chimaeric gene constructs into Arabidopsis cells, we showed that the A1 gene promoter mediates a transient expression about twofold higher than that obtained using the CaMV 35 S promoter. This expression depends on a 348 bp DNA fragment extending from -982 to -634 bp upstream of the initiation codon. This element contains a characteristic telomeric sequence (AACCCTAA) which is also found in the promoters of the A2 and A4 genes as well as in the promoters of the Drosophila EF-1 alpha F1 gene and of several highly expressed plant genes.

In vivo footprinting experiments have been used to analyze the binding of trans-acting regulatory factors in the 5' flanking region upstream of the alcohol dehydrogenase (Adh) gene from Arabidopsis thaliana. Protein-DNA interactions were detected by dimethyl sulfate footprinting and genomic sequencing, using an A. thaliana cell suspension culture that constitutively expressed the Adh gene. Several distinct footprinting domains have been characterized, and the potential effects of the corresponding trans-acting factors have been inferred from a comparison with data from the maize alcohol dehydrogenase-1 (Adh1) gene. One binding site is similar in sequence to one of the anaerobic response elements (ARE) of the maize gene, which has also been shown to bind to a trans-acting factor. Several of the remaining binding sites apparently represent a class of elements sharing the sequence 5'-GTGG-3' within their footprint. Comparisons with maize Adh1 in vivo protein interactions reveal that the elements of Adh promoter structure are highly conserved, but the relative and absolute positions of the elements are variable.

We are using the formation of trichomes in Arabidopsis thaliana as a model system to study gene expression during cellular differentiation. To initiate the molecular characterization of this system, we tagged and isolated a gene that is specifically required for the development of the specialized trichome cell. We confirmed the identity of this gene, GLABROUS1 (GL1), by complementation. These results demonstrate that a crucial gene in a plant developmental pathway can be successfully identified by complementation.

We have isolated a new complementation group of Arabidopsis thaliana long hypocotyl mutant (hy6) and have characterized a variety of light-regulated phenomena in hy6 and other previously isolated A. thaliana hy mutants. Among six complementation groups that define the HY phenotype in A. thaliana, three (hy1, hy2, and hy6) had significantly lowered levels of photoreversibly detectable phytochrome, although near wild-type levels of the phytochrome apoprotein were present in all three mutants. When photoregulation of chlorophyll a/b binding protein (cab) gene expression was examined, results obtained depended dramatically on the light regime employed. Using the red/far-red photoreversibility assay on etiolated plants, the accumulation of cab mRNAs was considerably less in the phytochrome-deficient mutants than in wild-type A. thaliana seedlings. When grown in high-fluence rate white light, however, the mutants accumulated wild-type levels of cab mRNAs and other mRNAs thought to be regulated by phytochrome. An examination of the light-grown phenotypes of the phytochrome-deficient mutants, using biochemical, molecular, and morphological techniques, revealed that the mutants displayed incomplete chloroplast and leaf development under conditions where wild-type chloroplasts developed normally. Thus, although phytochrome may play a role in gene expression in etiolated plants, a primary role for phytochrome in green plants is likely to be in modulating the amount of chloroplast development, rather than triggering the initiation of events (e.g., gene expression) associated with chloroplast development.

Interference with histidine metabolism, inhibition of pigment biosynthesis, or both have been the principal candidates for the primary site of action of 3-amino 1,2,4-triazole (amitrole). Arabidopsis thaliana is sensitive to 1,2,4-triazole-3-alanine, a feedback inhibitor of histidine biosynthesis, and this effect is reversed by histidine. The combination of triazolealanine and histidine, however, does not reverse the herbicidal effect of amitrole. This indicates that amitrole toxicity is not caused by histidine starvation, nor is it caused by the accumulation of a toxic intermediate of the histidine pathway. Amitrole inhibits root elongation at lower concentrations than it causes pigment bleaching in the leaves. In contrast, fluridone, a known inhibitor of the carotenoid biosynthetic pathway does not block root elongation. Fluridone also inhibits carotenoid accumulation in etiolated seedlings in the dark, but amitrole does not. Last, gabaculine and acifluorfen, but not amitrole, prevent chlorophyll accumulation in greening etiolated seedlings of Arabidopsis. These experiments cast doubt on pigment biosynthesis as the primary site of action of amitrole.

Mutants resistant to the herbicide N-(3-[1-ethyl-1-methylpropyl]-5-esoxazolyl)-2,6,dimethoxybenzamide (isoxaben) were recovered from an M2 population of Arabidopsis thaliana. Two of these mutants, DH47 and DH48, had a high level of resistance in the homozygous state. Crosses of these mutants to marker strains, and to each other, showed that each contained a mutation at a single locus tightly linked to lutescens, a marker on the fifth chromosome of A. thaliana. Growth curves of these mutants and of the F1 progeny of a cross with the wild type parent strain, in the presence of different concentrations of the herbicide, showed that both mutants display a semidominant phenotype. The two mutations differed in their degree of resistance, both as homozygotes and heterozygotes. This suggests that they are two different alleles. Callus cultures were established from plants homozygous, as well as heterozygous, for each of these mutations. Growth curves of these cultures in the presence of the herbicide mimicked the data obtained in vivo indicating that sensitivity to isoxaben is not dependent on a differentiated function.