Previously it was shown that transient chloramphenicol acetyltransferase (CAT) marker gene expression in Arabidopsis thaliana end Nicotiana tabacum resulted in significant differences in the accumulation of the CAT reaction products in radioactive CAT assays. Compared to Nicotiana tabacum, conversion of chloramphenicol to the acetylated products in Arabidopsis thaliana extracts was rather low. Here we report that the low CAT enzyme activity can be attributed in part to a heat sensitive CAT inhibitory effect in extracts of Arabidopsis thaliana. CAT enzyme activity in transgenic tobacco is inhibited by extracts from Arabidopsis. This inhibitory effect diminishes when Arabidopsis extracts were heat incubated. CAT activity in transgenic Arabidopsis lines was very low and was only detected in heat incubated extracts. Alternatively, enzyme-linked immunosorbent assays (ELISAs) can be used to detect the CAT protein in transgenic Arabidopsis.

The model dicotyledonous plant, Arabidopsis thaliana, is closely related to Brassica crop species. It is intended that information concerning the genetic control of basic biological processes in Arabidopsis will be transferable to other species. Genome collinearity and its potential to facilitate the identification of candidate genes in Arabidopsis homologous to genes controlling important agronomic traits in Brassica was investigated. Genetic mapping in B. nigra identified two loci influencing flowering time (FT), with loci on linkage groups 2 and 8 explaining 53% and 12% of the total variation in FT, respectively. The CO gene exerts an important control over FT in A. thaliana, and B. nigra homologues of CO probably also play an important role in regulating FT. B. nigra homologues of CO were identified on linkage groups 2 and 8, the homologue on group 2 was coincident with the major locus controlling FT while the homologue on group 8 was within the 90% confidence interval of the weaker FT gene. The CO homologue on group 2 exhibits abundant allelic variation suggesting that it naturally controls a wide range of flowering times. Fine-scale A. thaliana/B. nigra comparative mapping demonstrated short-range collinearity between the genomes of Arabidopsis and Brassica. Eleven DNA fragments spaced over a 1.5 Mb contig in A. thaliana were used as RFLP probes in B. nigra. Three collinear representations of the A. thaliana contig were identified in B. nigra, with one interrupted by a large chromosomal inversion. Collinearity over this range will allow the resources generated by the Arabidopsis genome project to facilitate map-based cloning in Brassica crops.

Flavonols are plant metabolites suggested to serve a vital role in fertilization of higher plants. Petunia and maize plants mutated in their flavonol biosynthesis are not able to set seed after self-pollination. We have investigated the role of these compounds in Arabidopsis thaliana. Like in all other plant species, high levels of flavonols could be detected in pollen of wild-type A. thaliana. No flavonols were detected in reproductive organs of the A. thaliana tt4 mutant in which the chs gene is mutated. Surprisingly, this mutant did set seed after self-fertilization and no pollen tube growth aberrations were observed in vivo. The role of flavonols during fertilization of Arabidopsis is discussed.

A cDNA clone was obtained from Arabidopsis thaliana that encodes a protein containing 92 amino acid residues with high sequence identity (57%) to bovine acyl-CoA-binding protein (ACBP). The coding sequence of this clone was expressed in Escherichia coli and the gene product (10.4 kDa) was purified. The recombinant A. thaliana ACBP (rAthACBP) was shown to bind acyl-CoA esters and protect acyl-CoAs from degradation by microsomal acyl-hydrolases. Antibodies that were raised to rAthACBP recognized the native Arabidopsis ACBP and also cross-reacted with a number of other plant ACBPs, including rapeseed (Brassica napus) ACBP. The pattern of expression and level of the gene product were examined in various tissues of Arabidopsis and Brassica using Western blotting. A. thaliana tissues contained between 3 and 143 micrograms AthACBP g-1 FW depending on the tissue (0.4 to 14 nmol g-1 FW). Developing B. napus seeds underwent a 12-fold increase in ACBP levels during seed maturation (20 to 250 micrograms ACBP g-1 FW); the highest concentration occurring near the peak of triacylglycerol accumulation (26 nmol g-1 FW).

A cDNA encoding a luminal binding protein (BiP) was isolated from Arabidopsis thaliana. In Arabidopsis seedlings, accumulation of transcripts for BiP was induced not only by inhibition of the N-glycosylation of proteins by tunicamycin but also by inhibition of the processing of N-linked glycans by castanospermine. Heat-shock stress also induced accumulation of the transcripts in Arabidopsis.

Endogenous brassinosteroids in the shoots of Arabidopsis thaliana were investigated. Castasterone, 6-deoxocastasterone, typhasterol and 6-deoxotyphasterol were identified by GC-MS. The co-occurrence of 6-deoxo-brassinosteroids and 6-oxo-brassinosteroids suggests that there are both early and late C6-oxidation pathways of brassinosteroids in A. thaliana.

A cDNA clone, pAUK1, with an open reading frame (ORF) coding for a hypothetical 164-amino-acid protein was isolated from an Arabidopsis thaliana (L.) Heynh cDNA library. The clone was attached, tail to tail, to the 3' end of A. thaliana hexokinase cDNA. An almost identical sequence had been previously described as the 5' untranslated region (5' UTR) of A. thaliana calmodulin cDNA (ACaM-2). Sequence comparison with three additional A. thaliana truncated cDNA clones which appear in a database (GenBank) supports the conclusion that pAUK1 is identical to the 5' UTR of ACaM-2 and that the 5'UTR of ACaM-2 is an independent cDNA artificially linked to A. thaliana calmodulin cDNA.

Using a PCR-amplified 5'-end proximal 600 bp fragment and two cDNA clones of cytosine DNA-methyltransferase gene of Arabidopsis thaliana as specific probes in hybridization with plant DNA samples, hydrolyzed by methylation-sensitive restriction endonucleases HpaII and MspI, it has been established that CCGG sites located in the 5'-end proximal part of cytosine DNA-methyltransferase gene are highly methylated at internal C and less but detectably methylated at external C residues. On the contrary, all CCGG sites in 3'-terminal half of the coding region were found to be unmethylated at both external and internal C residues. No significant differences between methylation patterns of cytosine DNA-methyltransferase gene in various organs (leaf, stem, flower) of the Arabidopsis thaliana plant were detected.

We have characterized the gravitropic response of inflorescence stems in Arabidopsis thaliana. When the inflorescence stems were placed horizontally, they curved upward about 90 degrees within 90 min in darkness at 23 degrees C, exhibiting strong negative gravitropism. Decapitated stem segments (without all flowers, flower buds, and apical apices) also showed gravitropic response when they included the elongation zone. This result indicates that the minimum elements needed for the gravitropic response exist in the decapitated inflorescence stem segments. At least the 3-min gravistimulation time was sufficient to induce the initial curvature at 23 degrees C after a lag time of about 30 min. In the gravitropic response of inflorescence stems, (a) the gravity perception site exists through the elongating zone, (b) auxin is involved in this response, (c) the gravitropic curvature was inhibited at 4 degrees C but at least the gravity perception step could occur, and (d) two curvatures could be induced in sequence at 23 degrees C by two opposite directional horizontal gravistimulations at 4 degrees C.