5-enolpyruvylshikimate-3-phosphate synthase (EPSPs), the target of the herbicide glyphosate, catalyzes an essential step in the shikimate pathway common to aromatic amino acid biosynthesis. We have cloned an EPSP synthase gene from Arabidopsis thaliana by hybridization with a petunia cDNA probe. The Arabidopsis gene is highly homologous to the petunia gene within the mature enzyme but is only 23% homologous in the chloroplast transit peptide portion. The Arabidopsis gene contains seven introns in exactly the same positions as those in the petunia gene. The introns are, however, significantly smaller in the Arabidopsis gene. This reduction accounts for the significantly smaller size of the gene as compared to the petunia gene. We have fused the gene to the cauliflower mosaic virus 35S promoter and reintroduced the chimeric gene into Arabidopsis. The resultant overproduction of EPSPs leads to glyphosate tolerance in transformed callus and plants.

High frequency transformation of Arabidopsis thaliana leaf explants has been obtained using a disarmed Ti plasmid containing the coding region of a neomycin phosphotransferase gene (NPT II) as a selectable marker. The rate of transformation ranged from 55 to 63 percent when acetosyringone (AS), a natural wound response molecule, was added to an Agrobacterium tumefaciens culture prior to incubation with leaf segments. Without acetosyringone, the transformation rate was approximately 2 to 3 percent. Calli resistant to G418 were regenerated into mature flowering plants in the presence of 10 micrograms/ml G418. Southern analysis and neomycin phosphotransferase assays confirmed the insertion and expression of the NPT II gene in regenerated Arabidopsis plants.

Germination and growth of wild-type and two mutant strains (aux-1 and Dwf) of Arabidopsis thaliana L. have been examined. Seedlings of aux-1 exhibit agravitropic roots whereas Dwf display both agravitropic roots and shoots. Wild-type seedlings retained the seed coat at the root-hypocotyl transition zone and developed hypocotyl hooks. In contrast, aux-I and Dwf seedlings did not retain their seed coats and lacked hypocotyl hooks. A positive gravitropic response of the roots was essential for the retention of the seed coat at the root—hypocotyl transition zone by the attachment of root hairs to the seed coat. The development of the hypocotyl hook was aided by the retention of the seed coat. The apical region of the hypocotyl apparently remained agravitropic during formation and maintenance of the hypocotyl hook.

We have characterized the beta-tubulin gene family of Arabidopsis thaliana. Five distinct genes were cloned. and analyzed by restriction enzyme mapping and cross-hybridization studies. Three of the genes appear to be dispersed, whereas two others are linked within 1.5 kb of one another. The two linked genes are closely related and appear to have resulted from a fairly recent duplication. The three dispersed genes do not cross-hybridize to one another or to the two linked genes under highly stringent hybridization conditions, suggesting that they arose from more ancient duplications. From Southern analysis we estimate that there are a total of between six and ten beta-tubulin genes in Arabidopsis. Additional analyses indicate that the gene family is equal in size or larger than those in other plants, but significantly smaller than those in related Brassica species. Sequence determination of one of the Arabidopsis genes revealed a highly unusual transcribed leader sequence. The leader contains two fairly long tracks of adenines. One is located toward the 5' end of the mRNA and the other is just before the initiation codon. A track of uridines is located between the adenine tracks. This leader can form two different secondary structures that may have regulatory significance.