A new mode of herbicidal action was established by finding specific inhibitors of imidazoleglycerol phosphate dehydratase, an enzyme of histidine (His) biosynthesis. Three triazole phosphonates inhibited the reaction of the enzyme with Ki values of 40 f 6.5, 10 f 1.6, and 8.5 2 1.4 nM, respectively, and were highly cytotoxic to cultured plant cells. This effect was completely reversed by the addition of His, proving that the cytotoxicity was primarily caused by the inhibition of His biosynthesis. These inhibitors showed widespectrum, postemergent herbicidal activity at application rates ranging from 0.05 to 2 kg/ha.

cDNA clones encoding imidazoleglycerolphosphate dehydratase (ICPD; EC 4.2.1.19) from Arabidopsis thaliana were isolated by complementation of a bacterial auxotroph. The predicted primary translation product shared significant identity with the corresponding sequences from bacteria and fungi. As in yeast, the plant enzyme is monofunctional, lacking the histidinol phosphatase activity present in the Escherichia coli protein. ICPD mRNA was present in major organs at ali developmental stages assayed. The Arabidopsis genome appears to contain two genes encoding this enzyme, based on DNA gel blot and polymerase chain reaction analysis.

The partial amino acid sequence of histidinol dehydrogenase (L-histidinol:NAD+ oxidoreductase, EC 1.1.1.23) from cabbage was determined from peptide fragments of the purified protein. The relative positions of these peptides were deduced by aligning their sequences with the sequence of the HIS4C gene product of Saccharomyces cerevisiae. cDNA encoding histidinol dehydrogenase was then amplified from a library using a polymerase chain reaction primed with degenerate oligonucleotide pools of known position and orientation. By using this amplified fragment as a probe, an apparently fulD-length cDNA clone was isolated that is predicted to encode a proenzyme having a putative 31-amino acid chloroplast transit peptide and a mature molecular mass of47.5 kDa. The predicted protein sequence was 51% identical to the yeast enzyme and 49% identical to the Escherichia coli enzyme. Expression of the cDNA clone in an E. coli his operon deletion strain rendered the mutant able to grow in the presence of histidinol.In plants, the biosynthetic pathways of most of the amino acids are poorly understood. Only a handful of enzymes involved in amino acid biosynthesis have been purified from plant sources, partly because of the small amount of these proteins found in plant cells. To date, glutamate synthase, glutamate dehydrogenase, and glutamine synthetase in the glutamate pathway (1-3), aspartate kinase (4) and homoserine dehydrogenase (5) in the threonine pathway, dihydrodipicolinate synthase (6) in the lysine pathway, and 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (7) and 5-enoylpyruvyl shikimate-3-phosphate synthase (8) in the aromatic amino acid pathway have been characterized and purified from various plant species. cDNAs encoding several of these enzymes have been cloned (8-12), as have genes for other amino acid biosynthetic enzymes based on their structural or functional homology to microbial or mammalian enzymes (13-17).Although histidine biosynthesis has been elucidated in several prokaryotic and eukaryotic microbes (18-22), the biosynthetic pathway in higher plants is unknown (23). Indirect evidence has indicated that the pathway follows a route similar to that found in bacteria and fungi (24,25). Recently, histidinol dehydrogenase (HDH; L-histidinol:NAD+ oxidoreductase, EC 1.1.1.23) was purified to homogeneity from Brassica oleracea (cabbage; ref. 26), proving that the final steps in histidine biosynthesis proceed in plants as they do in prokaryotes and fungi.In this report, we describe the isolation ofa full-length cDNA encoding HDH from cabbage. The plant coding sequence ¶ was approximately 50o identical to known microbial genes. To demonstrate function of the plant clone, the cDNA was expressed in an Escherichia coli strain lacking the histidine operon. The cDNA was found to suppress the his deletion when the bacteria were grown in the presence of histidinol. MATERIALS AND METHODSPlant Material and Bacterial Strains. Mature spring cabbage (Brassica oleracea L. var capitata L.) was purchased from a local...

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lmidazoleglycerolphosphate dehydratase (ICPD; EC 4.2.1.1 9), which is involved in the histidine biosynthetic pathway of Arabidopsis fhaliana and wheat (Triticum aestivum), has been expressed in insect cells using the baculovirus expression vector system. Nterminal amino acid sequencing indicated that recombinant IGPDs (rlCPDs) were produced as mature forms via nonspecific proteolytic cleavages in the putative transit peptide region. The wheat rlCPD contained one M n atom per subunit, and the M n was involved in the assembly of the subunits to form active ICPDs. Protein-blotting analysis, using antibodies raised against the wheat rlCPD, indicated that ICPD was located in the chloroplasts of wheat. The rlCPDs of Arabidopsis and wheat, which were 86% identical in their primary structures deduced from the cDNAs, exhibited similar properties in terms of the molecular mass, pH optimum, and the K,,, for the substrate, imidazoleglycerolphosphate. However, the nonselective herbicides 3-amino-l,2,4-triazole and a newly synthesized triazole [(l R', 3Re)-[3-hydroxy-3-(2H-[1,2,4]triazole-3-yl)-cyclohexyl]-phosphonic acid], inhibited Arabidopsis and wheat ICPDs in a mixed-type and a competitive manner, respectively.